JP3614183B2 - Reconstituted human antibody against human interleukin-6 - Google Patents

Description

【００４０】
マウスＳＫ２のＬ鎖Ｖ領域はヒトＬ鎖Ｖ領域のサブグループＩ（ＨＳＧＩ）のコンセンサス配列に最も類似しており、６０．３％の相同性が存在する。マウスＳＫ２のＨ鎖Ｖ領域はヒトＨ鎖Ｖ領域のサブグループＩＩ（ＨＳＧＩＩ）のコンセンサス配列に最も類似しており、５９．２％の相同性が存在する。
【００４１】
ヒト抗体中のＶ領域との比較から、再構成ヒトＳＫ２抗体Ｖ領域の設計の基礎となるヒトＶ領域のＦＲを選択することが可能である。再構成ヒトＳＫ２抗体Ｌ鎖Ｖ領域の設計のためにはサブグループＩ（ＨＳＧＩ）に属するヒトＬ鎖Ｖ領域を使用し、そして再構成ヒトＳＫ２抗体Ｈ鎖Ｖ領域の設計のためにはサブグループＩＩ（ＨＳＧＩＩ）に属するヒト抗体Ｈ鎖Ｖ領域を用いるのがよく、さらにサブグループ内で、できるだけ相同性の高いものを選ぶことが最善であろう（Ｋａｂａｔ，Ｅ．Ａ．ら、ＵＳ Ｄｅｐ．Ｈｅａｌｔｈ ａｎｄ Ｈｕｍａｎ Ｓｅｒｖｉｃｅｓ，ＵＳ Ｇｏｖｅｒｎｍｅｎｔ Ｐｒｉｎｔｉｎｇ Ｏｆｆｉｃｅｓ，１９９１）。
【００４２】
再構成ヒトＳＫ２抗体Ｖ領域の設計
再構成ヒトＳＫ２抗体Ｖ領域の設計における第一段階は、設計の基礎となるヒト抗体Ｖ領域を選択することであった。マウスＳＫ２抗体Ｌ鎖Ｖ領域は、サブグループＩに属するヒトκ型抗体Ｌ鎖Ｖ領域に最も類似していた（表１）。マウスＳＫ２抗体のＬ鎖Ｖ領域は既知ヒト抗体Ｌ鎖Ｖ領域との比較において、ヒトＬ鎖Ｖ領域のサブグループＩの１構成員であるヒトＬ鎖Ｖ領域ＲＥＩに６３％の類似性を示した。従って、再構成ヒトＳＫ２抗体Ｌ鎖Ｖ領域の作製のための出発材料としてＲＥＩのＦＲを使用した。
【００４３】
再構成ヒトＳＫ２抗体Ｌ鎖Ｖ領域の２つのバージョンを設計した。第一のバージョン（バージョン「ａ」）においては、ヒトＦＲは再構成ヒトＣＡＭＰＡＴＨ−１Ｈ抗体中に存在するＲＥＩに基くＦＲ（Ｒｉｅｃｈｍａｎｎ，Ｌ．ら、Ｎａｔｕｒｅ ３３２，３２３〜３２７，１９８８）（ＷＯ９２−１９７５９に記載の再構成ヒトＰＭ−１のＬ鎖Ｖ領域のバージョン「ａ」）と同一であり、そしてマウスＣＤＲはマウスＳＫ２のＬ鎖Ｖ領域中のＣＤＲと同一とした。第二のバージョン（バージョン「ｂ」）はバージョン「ａ」と比べて、ヒトＦＲ３中の位置７１におけるアミノ酸１個のみを異にする。
【００４４】
すなわちバージョン「ａ」の設計に使用した修飾されたＲＥＩのＦＲにおいては位置７１はフェニルアラニンであるのに対し、バージョン「ｂ」においては、位置７１のフェニルアラニンがマウスＳＫ２抗体Ｌ鎖Ｖ領域中に見出されるようにチロシンに変えられている。表２は、マウスＳＫ２抗体のＬ鎖Ｖ領域、ＲＥＩのＦＲ及び再構成ＳＫ２抗体のＬ鎖Ｖ領域の２種類のバージョンの、それぞれのアミノ酸配列を示す。
【００４５】
【表２】

注：ＲＥＩのＦＲ中の５個の下線を付したアミノ酸はヒトＲＥＩのアミノ酸配列（Ｐａｌｍ，Ｗ．ら、Ｈｏｐｐｅ−Ｓｅｙｌｅｒ’ｓ，Ｚ．Ｐｈｙｓｉｏｌ．Ｃｈｅｍ．，３５６，１６７−１９１，１９７５）とは異なるアミノ酸の位置を示す。
【００４６】
マウスＳＫ２抗体のＨ鎖Ｖ領域中のＦＲはサブグループＩＩに属するヒトＨ鎖Ｖ領域に最も類似している（表１）。マウスＳＫ２抗体のＨ鎖Ｖ領域は既知のヒト抗体Ｈ鎖Ｖ領域との比較において、ヒトＨ鎖Ｖ領域のサブグループＩＩの１構成員であるヒトＨ鎖Ｖ領域ＤＡＷ（Ｐｒｅｓｓ，Ｅ．Ｍ．ら、Ｂｉｏｃｈｅｍ．Ｊ．１１７，６４１，１９７０）に非常に類似していた（６０．８％）。さらには、ＣＤＲのサイズもマウスＳＫ２抗体とヒト抗体ＤＡＷとの間で非常に類似していた。このため、ヒト抗体ＤＡＷのＦＲを、再構成ヒトＳＫ２抗体のＨ鎖Ｖ領域の作製のための出発材料として用いた。
【００４７】
再構成ヒトＳＫ２抗体Ｈ鎖Ｖ領域の２種類のバージョンを設計した。バージョン「ａ」においてはＦＲはＤＡＷのそれと同じであり、バージョン「ｂ」においてはヒトＦＲ１中の３０位のみが異っている。すなわち、バージョン「ａ」においては３０位のアミノ酸はセリンであり、バージョン「ｂ」の３０位のアミノ酸はアスパラギンである。
表３は、マウスＳＫ２抗体Ｈ鎖Ｖ領域、ＤＡＷのＦＲ、並びに再構成ＳＫ２抗体Ｈ鎖Ｖ領域のバージョン「ａ」及び「ｂ」のアミノ酸配列を示す。
【００４８】
【表３】

【００４９】
再構成ヒトＳＫ２抗体の作製
再構成ヒトＳＫ抗体Ｖ領域の作製を実施例５に具体的に記載する。
本発明の再構成ヒトＳＫ２抗体は、
（Ａ）（１）ヒトＬ鎖Ｃ領域、及び
（２）ヒトＬ鎖ＦＲ、及びヒトＩＬ−６に対するマウスモノクローナル抗体ＳＫ２のＬ鎖Ｖ領域ＣＤＲを含んで成るＬ鎖Ｖ領域、を含んで成るＬ鎖；並びに
（Ｂ）（１）ヒトＨ鎖Ｃ領域、及び
（２）ヒトＨ鎖ＦＲ、及びヒトＩＬ−６に対するマウスモノクローナル抗体ＳＫ２のＨ鎖Ｖ領域ＣＤＲを含んで成るＨ鎖Ｖ領域、を含んで成るＨ鎖；
から構成される。
【００５０】
好ましい態様においては、前記Ｌ鎖Ｖ領域は配列番号５２又は５３に示されるアミノ酸配列を有し、前記Ｈ鎖Ｖ領域は配列番号５４又は５５に示されるアミノ酸配列を有する。前記ヒトＬ鎖Ｃ領域は任意のヒトＬ鎖Ｃ領域であることができ、例えばヒトκＣ領域であり；そして前記ヒトＨ鎖Ｃ領域は任意のヒトＨ鎖Ｃ領域であることができ、例えばヒトγ−１Ｃ領域、ヒトγ−４Ｃ領域等である。
【００５１】
再構成抗体の製造のためには、２種類の発現ベクター、すなわちエンハンサー／プロモーター系のごとき発現制御領域による制御のもとに前に定義した再構成ヒトＬ鎖をコードするＤＮＡを含んで成る発現ベクター、及びエンハンサー／プロモーター系のごとき発現制御領域のもとに前に定義した再構成ヒトＨ鎖をコードするＤＮＡを含んで成るもう一つの発現ベクターを作製する。次に、これらの発現ベクターを用いて哺乳類細胞のごとき宿主細胞を同時形質転換し、そしてこの形質転換された細胞をイン−ビボ又はイン−ビトロで培養して再構成ヒト抗体を生産せしめる（例えば、ＷＯ９１−１６９２７）。
【００５２】
あるいは、再構成Ｌ鎖をコードするＤＮＡ及び再構成ヒトＨ鎖をコードするＤＮＡを単一の発現ベクターに導入し、そしてこのベクターを用いて宿主を形質転換し、次にこの形質転換された宿主細胞をイン−ビボ又はイン−ビトロで培養して目的とする再構成ヒト抗体を生産せしめる。
以上のようにして目的とするキメラ抗体あるいはヒト型化抗体をコードする遺伝子で形質転換した形質転換体を培養し、産生したキメラ抗体あるいはヒト型化抗体は、細胞内または細胞外から分離し均一にまで精製することができる。
【００５３】
なお、本発明の目的蛋白質であるキメラ抗体あるいはヒト型化抗体の分離・精製を、プロテインＡアガロースカラムを用いて行うことができる。また、その他に、通常の蛋白質で用いられる分離・精製方法を使用すればよく、何ら限定されるものではない。例えば各種クロマトグラフィー、限外濾過、塩折、透析等を適宜選択、組合せれば、キメラ抗体あるいはヒト型化抗体は分離・精製することができる。
【００５４】
ヒトＩＬ−６に対する本発明のキメラ抗体又は再構成ヒト抗体の製造のために任意の発現系、例えば真核細胞、例えば動物細胞、例えば樹立された哺乳類細胞系、真糸状菌細胞、及び酵母細胞、並びに原核細胞、例えば細菌細胞、例えば大腸菌細胞等を使用することができる。好ましくは、本発明のキメラ抗体又は再構成抗体は哺乳類細胞、例えばＣＯＳ細胞又はＣＨＯ細胞中で発現される。
【００５５】
これらの場合、哺乳類細胞での発現のために有用な常用のプロモーターを用いることができる。例えば、ヒト・サイトメガロウィルス前期（ｈｕｍａｎ ｃｙｔｏｍｅｇａｌｏｖｉｒｕｓ ｉｍｍｅｄｉａｔｅ ｅａｒｌｙ；ＨＣＭＶ）プロモーターを使用するのが好ましい。ＨＣＭＶプロモーターを含有する発現ベクターの例には、ＨＣＭＶ−Ｖ_Ｈ −ＨＣγ１，ＨＣＭＶ−Ｖ_Ｌ −ＨＣ_Ｋ 等であって、ｐＳＶ２ｎｅｏに由来するもの（図２を参照のこと）（ＷＯ９２−１９７５９）が含まれる。
【００５６】
また、その他に、本発明のために用いることのできる哺乳動物細胞における遺伝子発現のプロモーターとしてはレトロウイルス、ポリオーマウイルス、アデノウイルス、シミアンウイルス４０（ＳＶ４０）などのウイルスプロモーターやヒト・ポリペプチド・チェーン・エロンゲーション・ファクター１α（ＨＥＦ−１α）などの哺乳動物細胞由来のプロモーターを用いればよい。例えばＳＶ４０のプロモーターを使用する場合は、Ｍｕｌｌｉｇａｎらの方法（Ｎａｔｕｒｅ ２７７ １０８（１９７９））、また、ＨＥＦ−１αプロモーターを使用する場合は、Ｍｉｚｕｓｈｉｍａ，Ｓ．らの方法（Ｎｕｃｌｅｉｃ Ａｃｉｄｓ Ｒｅｓｅａｒｃｈ，１８，５３２２，１９９０）に従えば容易に実施することができる。
【００５７】
複製起原としては、ＳＶ４０、ポリオーマウイルス、アデノウイルス、牛パピローマウイルス（ＢＰＶ）等の由来のものを用いることができ、さらに宿主細胞系中での遺伝子コピー数増幅のため、発現ベクターは選択マーカーとして、ホスホトランスフェラーゼＡＰＨ（３′）ＩＩあるいはＩ（ｎｅｏ）遺伝子、チミジンキナーゼ（ＴＫ）遺伝子、大腸菌キサンチン−グアニンホスホリボシルトランスフェラーゼ（Ｅｃｏｇｐｔ）遺伝子、ジヒドロ葉酸還元酵素（ＤＨＦＲ）遺伝子等を含むことができる。
【００５８】
【実施例】
次に、本発明を下記の実施例により具体的に説明するが、これにより本発明の範囲が限定されるものではない。
実施例１．ヒトＩＬ−６に対するマウスモノクローナル抗体のＶ領域をコードするＤＮＡのクローン化
ヒトＩＬ−６に対するマウスモノクローナル抗体ＳＫ２の可変領域をコードするＤＮＡを次の様にしてクローン化した。
【００５９】
１．全ＲＮＡの調製
ハイブリドーマＳＫ２からの全ＲＮＡを、Ｃｈｉｒｇｗｉｎら、Ｂｉｏｃｈｅｍｉｓｔｒｙ，１８，５２９４（１９７９）により記載されている方法に従って調製した。すなわち、ハイブリドーマＳＫ２の細胞を２０ｍｌの４Ｍグアニジンチオシアネート（Ｆｕｌｋａ）中で完全にホモジナイズさせた。ホモジネートを遠心管中の５．３Ｍ塩化セシウム溶液層上に重層し、次にこれをＢｅｃｋｍａｎ ＳＷ４０ローター中で３１，０００ｒｐｍ にて２０℃で２４時間遠心分離することによりＲＮＡを沈澱させた。
【００６０】
ＲＮＡ沈澱物を８０％エタノールにより洗浄し、そして１ｍＭ ＥＤＴＡ及び０．５％ ＳＤＳを含有する１０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．５）１５０μｌ中に溶解し、そしてそれにＰｒｏｔｅｉｎａｓｅ（Ｂｏｅｈｒｉｎｇｅｒ）を０．５ｍｇ／ｍｌとなるように添加した後、３７℃にて２０分間インキュベートした。混合物をフェノール及びクロロホルムで抽出し、そしてＲＮＡをエタノールで沈澱させた。次に、ＲＮＡ沈澱物を１ｍＭ ＥＤＴＡを含有する１０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．５）２００μｌに溶解した。
【００６１】
２．一本鎖ｃＤＮＡの合成
Ｊ．Ｗ．Ｌａｒｒｉｃｋら、Ｂｉｏ／Ｔｅｃｈｎｏｌｏｇｙ，７，９３４（１９８９）により記載されている方法に従って一本鎖ｃＤＮＡを合成するため、前記のようにして調製した全ＲＮＡの約５μｇを４０ｍＭ ＫＣｌ，６ｍＭ ＭｇＣｌ_２ 、１０ｍＭジチオスレイトール、０．５ｍＭ ｄＡＴＰ，０．５ｍＭ ｄＧＴＰ，０．５ｍＭ ｄＣＴＰ，０．５ｍＭ ｄＴＴＰ，３５μＭ ｏｌｉｇｏ ｄＴプライマー（Ａｍｅｒｓｈａｍ）、４８ユニットのＲＡＶ−２逆転写酵素（ＲＡＶ−２：Ｒｏｕｓ ａｓｓｏｃｉａｔｅｄ ｖｉｒｕｓ２；Ａｍｅｒｓｈａｍ）及び２５ユニットのヒト胎盤リボヌクレアーゼ阻害剤（Ａｍｅｒｓｈａｍ）を含有する５０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．３）緩衝液１０μｌに溶解し、そしてこの反応混合物を３７℃にて６０分間インキュベートしそして次のポリメラーゼ連鎖反応（ＰＣＲ）法のために直接使用した。
【００６２】
３．抗体可変領域をコードする遺伝子のＰＣＲ法による増幅
Ｔｈｅｒｍａｌ Ｃｙｃｌｅｒ Ｍｏｄｅｌ ＰＨＣ−２（Ｔｅｃｈｎｅ社）を用いてＰＣＲ法を行った。
（１）マウスＬ鎖Ｖ領域をコードする遺伝子の増幅
ＰＣＲ法に使用するプライマーは、マウスカッパ型Ｌ鎖リーダー配列とハイブリダイズする配列番号：１〜１１に示すＭＫＶ（Ｍｏｕｓｅ Ｋａｐｐａ Ｖａｒｉａｂｌｅ）プライマー（Ｓ．Ｔ．Ｊｏｎｅｓら、Ｂｉｏ／Ｔｅｃｈｎｏｌｏｇｙ，９，８８，１９９１）、及びマウスκ型Ｌ鎖Ｃ領域とハイブリダイズする配列番号：１２に示すＭＫＣ（Ｍｏｕｓｅ Ｋａｐｐａ Ｃｏｎｓｔａｎｔ）プライマー（Ｓ．Ｔ．Ｊｏｎｅｓら、Ｂｉｏ／Ｔｅｃｈｎｏｌｏｇｙ，９，８８，１９９１）を用いた。
【００６３】
ＰＣＲ溶液１００μｌは、１０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．３），５０ｍＭ ＫＣｌ，０．２５ｍＭ ｄＮＴＰｓ（ｄＡＴＰ，ｄＧＴＰ，ｄＣＴＰ，ｄＴＴＰ）、１．５ｍＭ ＭｇＣｌ_２ 、２．５ユニットのＤＮＡポリメラーゼＡｍｐｌｉＴａｑ（Ｐｅｒｋｉｎ Ｅｌｍｅｒ Ｃｅｔｕｓ社）、０．２５μＭのそれぞれのＭＫＶプライマー、２．７５μＭのＭＫＣプライマー及び一本鎖ｃＤＮＡ合成の反応混合物１μｌを含有する。これを５０μｌの鉱油で覆った後、９４℃の初期温度にて１．５分間そして次に９４℃にて１分間、５０℃にて１分間及び７２℃にて１分間、この順序で加熱した。この温度サイクルを２５回反復した後、反応混合物をさらに７２℃にて１０分間インキュベートした。
【００６４】
（２）マウスＨ鎖Ｖ領域をコードするｃＤＮＡの増幅
ＰＣＲのためのプライマーとして配列番号：１３〜２４に示すＭＨＶ（Ｍｏｕｓｅ Ｈｅａｖｙ Ｖａｒｉａｂｌｅ）プライマー１〜１２、及び配列番号：２５に示すＭＨＣ−Ｇ１（Ｍｏｕｓｅ Ｈｅａｖｙ Ｃｏｎｓｔａｎｔ）プライマー（Ｓ．Ｔ．Ｊｏｎｅｓら、Ｂｉｏ／Ｔｅｃｈｎｏｌｏｇｙ，９，８８，１９９１）を使用した。
【００６５】
ｃＤＮＡの増幅は、１μＭのＭＨＶプライマーと１μＭのＭＨＣ−Ｇ１プライマーの組合せとして各々のＭＨＶ１〜１２プライマーについて別々に増幅した点を除いて、前記３．（１）においてＬ鎖Ｖ領域遺伝子の増幅について記載したのと同じ方法により増幅を行った。
【００６６】
４．ＰＣＲ生成物の精製および断片化
前記のようにしてＰＣＲ法により増幅したＤＮＡ断片を低融点アガロース（ＦＭＣ Ｂｉｏ．Ｐｒｏｄｕｃｔｓ，米国）にて精製し、そして１０ｍＭ ＭｇＣｌ_２ 及び１５０ｍＭ ＮａＣｌを含有する１００ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．６）中で１０ユニットの制限酵素ＳａｌＩ（ＧＩＢＣＯ ＢＲＬ社製）を用いて３７℃にて３時間消化した。
【００６７】
この消化混合物をフェノール及びクロロホルムで抽出し、そしてＤＮＡをエタノール沈澱により回収した。次に、ＤＮＡ沈澱物を１０ユニットの制限酵素ＸｍａＩ（Ｎｅｗ Ｅｎｇｌａｎｄ Ｂｉｏｌａｂｓ社製）により３７℃にて２時間消化し、そして生ずるＤＮＡ断片を、１．５％低融点アガロース（ＦＭＣ Ｂｉｏ．Ｐｒｏｄｕｃｔｓ，米国）を用いるアガロースゲル電気泳動により分離した。
【００６８】
約４５０ｂｐ長のＤＮＡ断片を含有するアガロース片を切り取りそして６５℃にて５分間溶融せしめ、そしてこれと同容積の２ｍＭ ＥＤＴＡ及び２００ｍＭ ＮａＣｌを含有する２０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．５）を加えた。この混合物をフェノール及びクロロホルムにより抽出し、そしてＤＮＡ断片をエタノール沈澱により回収し、そして１ｍＭ ＥＤＴＡを含有する１０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．５）に溶解した。
【００６９】
こうして、マウスカッパ型Ｌ鎖可変領域をコードする遺伝子を含んで成るＤＮＡ断片、及びマウスＨ鎖可変領域をコードする遺伝子を含んで成るＤＮＡ断片を得た。上記ＤＮＡ断片はいずれもその５′−末端にＳａｌＩ接着末端を有しそしてその３′−末端にＸｍａＩ接着末端を有する。
【００７０】
５．連結及び形質転換
上記のようにして調製したマウスカッパ型Ｌ鎖Ｖ領域をコードする遺伝子を含んで成るＳａｌＩ−ＸｍａＩ ＤＮＡ断片約０．３μｇを、ＳａｌＩ及びＸｍａＩで消化することにより調製したｐＵＣ１９ベクター約０．１μｇと、５０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．４），１０ｍＭ ＭｇＣｌ_２ 、１０ｍＭジチオスレイトール、１ｍＭスペルミジン、１ｍＭ ＡＴＰ，０．１μｇ／ｍｌのウシ血清アルブミン及び２ユニットＴ４ ＤＮＡリガーゼ（Ｎｅｗ Ｅｎｇｌａｎｄ Ｂｉｏｌａｂｓ）を含有する反応混合物中で、１６℃にて１６時間反応させ連結した。
【００７１】
次に、７μｌの上記連結混合物を大腸菌ＤＨ５αのコンピテント細胞２００μｌに加え、そしてこの細胞を氷上で３０分間、４２℃にて１分間そして再び氷上で１分間静置した。次いで８００μｌのＳＯＣ培地（Ｍｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ：Ａ Ｌａｂｏｒａｔｏｒｙ Ｍａｎｕａｌ，Ｓａｍｂｒｏｏｋら、Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ Ｐｒｅｓｓ，１９８９）を加え、３７℃にて１時間インキュベートした後、２×ＹＴ寒天培地（Ｍｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ：Ａ Ｌａｂｏｒａｔｏｒｙ Ｍａｎｕａｌ，Ｓａｍｂｒｏｏｋら、Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ Ｐｒｅｓｓ，１９８９）上にこの大腸菌をまき、３７℃にて一夜インキュベートして大腸菌形質転換体を得た。
【００７２】
この形質転換体を、５０μｇ／ｍｌのアンピシリンを含有する２×ＹＴ培地５ｍｌ中で３７℃にて一夜培養し、そしてこの培養物から、アルカリ法（Ｍｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ：Ａ Ｌａｂｏｒａｔｏｒｙ Ｍａｎｕａｌ，Ｓａｍｂｒｏｏｋら、Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ Ｐｒｅｓｓ，１９８９）に従ってプラスミドＤＮＡを調製した。
【００７３】
こうして得られた、ハイブリドーマＳＫ２に由来するマウスカッパ型Ｌ鎖Ｖ領域をコードする遺伝子を含有するプラスミドをｐｓｋ２−ｋ２と命名した。
上記の同じ方法に従って、ハイブリドーマＳＫ２に由来するマウスＨ鎖Ｖ領域をコードする遺伝子を含有するプラスミドをＳａｌＩ−ＸｍａＩ ＤＮＡ断片から作成し、そしてｐＵＣ−ＳＫ２−Ｖ_Ｈ と命名した。
【００７４】
実施例２．ＤＮＡの塩基配列の決定
前記のプラスミド中のｃＤＮＡコード領域の塩基配列を、Ｓｅｑｕｅｎａｓｅ^ＴＭＶｅｒｓｉｏｎ２．０キット（Ｕ．Ｓ．Ｂｉｏｃｈｅｍｉｃａｌ 社製，米国）を用いて決定した。
まず、前記のようにして得られたプラスミド約３μｇを０．２Ｎ ＮａＯＨにより変性し、配列決定用プライマーとアニールさせ、そしてキット添付の処方に従って^３５Ｓ−ｄＡＴＰより標識した。
【００７５】
次に、標識されたＤＮＡを、８Ｍ尿素を含有する６％ポリアクリルアミドゲルにて電気泳動した後、ゲルを１０％メタノール及び１０％酢酸により固定し、乾燥し、そしてオートラジオグラフィーにかけることにより塩基配列を決定した。
プラスミドｐｓｋ２−ｋ２に含まれるマウスＳＫ２抗体のＬ鎖Ｖ領域をコードする遺伝子の塩基配列を配列番号２６に示す。また、プラスミドｐＵＣ−ＳＫ２−Ｖ_Ｈ に含まれるマウスＳＫ２抗体のＨ鎖Ｖ領域をコードする遺伝子の塩基配列を配列番号２７に示す。
【００７６】
なお、前記プラスミドｐＵＣ−ＳＫ２−Ｖ_Ｈ を含有する大腸菌はＥｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ ＤＨ５α（ｐＵＣ−ＳＫ２Ｖ_Ｈ ）として、工業技術院生命工学工業技術研究所（茨城県つくば市東１丁目１番３号）に、平成５年４月２１日に、ＦＥＲＭ ＢＰ−４２６９としてブダペスト条約に基き国際寄託された。
【００７７】
実施例３．ＣＤＲの決定
Ｌ鎖及びＨ鎖のＶ領域の全般的構造は、互いに類似性を有しており、それぞれ４つのフレームワーク部分が３つの超可変領域、即ち相補性決定領域（ＣＤＲ）により連結されている。フレームワークのアミノ酸配列は、比較的良く保存されているが、一方、ＣＤＲ領域のアミノ酸配列の変異性は極めて高い（Ｋａｂａｔ，Ｅ．Ａ．ら、「Ｓｅｑｕｅｎｃｅｓ ｏｆ Ｐｒｏｔｅｉｎｓ ｏｆ Ｉｍｍｕｎｏｌｏｇｉｃａｌ Ｉｎｔｅｒｅｓｔ」ＵＳ Ｄｅｐｔ．Ｈｅａｌｔｈ ａｎｄ Ｈｕｍａｎ Ｓｅｒｖｉｃｅｓ，１９８３）。
【００７８】
この様な事実に基づき、ヒトＩＬ−６に対するマウスモノクローナル抗体の可変領域のアミノ酸配列をＫａｂａｔらにより作成された抗体のアミノ酸配列のデータベースにあてはめて、相同性を調べることによりＣＤＲ領域を表４に示す如く決定した。
【表４】

【００７９】
実施例４．クローン化ｃＤＮＡの発現の確認（キメラＳＫ２抗体の作製）
発現ベクターの作製
キメラＳＫ２抗体を発現するベクターを作製するため、それぞれマウスＳＫ２κＬ鎖及びＨ鎖Ｖ領域をコードするｃＤＮＡクローンｐｓｋ２−ｋ２及びｐＵＣ−ＳＫ２−Ｖ_Ｈ をＰＣＲ法により修飾した。そしてＨＥＦ発現ベクター（前記、ＷＯ９２−１９７５９参照）（図１を参照のこと）に導入した。
【００８０】
Ｌ鎖Ｖ領域のための後方プライマーＳＫ２Ｋ２Ｂ（配列番号：２８）及びＨ鎖Ｖ領域のための後方プライマーＳＫ２Ｈ１Ｓ（配列番号：３０）は、各々のＶ領域のリーダー配列の最初をコードするＤＮＡにハイブリダイズし且つＫｏｚａｋコンセンサス配列（Ｋａｚａｋ，Ｍ．ら、Ｊ．Ｍｏｌ．Ｂｉｏｌ．１９６，９４７−９５０，１９８７）及びＨｉｎｄＩＩＩ 制限部位を有するように設計した。Ｌ鎖Ｖ領域のための前方プライマーＳＫ２Ｋ２Ａ（配列番号：２９）及びＨ鎖Ｖ領域のための前方プライマー６４ｈｃｈ−Ａ（配列番号：３１）は、Ｊ領域の末端をコードするＤＮＡ配列にハイブリダイズし且つスプライスドナー配列及びＢａｍＨＩ制限部位を有するように設計した。
【００８１】
１０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．３），５０ｍＭ ＫＣｌ，２５０μＭ ｄＮＴＰｓ，１．５ｍＭ ＭｇＣｌ_２ 、１００ｐｍｏｌｅ ずつの各プライマー、１００ｎｇの鋳型ＤＮＡ（ｐｓｋ２−ｋ２又はｐＵＣ−ＳＫ２−Ｖ_Ｈ ）、及び５ｕのＡｍｐｌｉ Ｔａｑ酵素を含有する１００μｌのＰＣＲ反応混合物を５０μｌの鉱油で覆い、９４℃にて最初の変性の後、９４℃にて１分間、５０℃にて１分間、７２℃にて１分間のサイクルを３０回行い、最後に７２℃にて１０分間インキュベートした。
【００８２】
ＰＣＲ生成物を１．５％低融点アガロースゲルを用いて精製し、ＨｉｎｄＩＩＩ 及びＢａｍＨＩで消化し、そしてｐＵＣ１９ベクター（Ｙａｎｉｓｈｅ−Ｐｅｒｒｏｎら、Ｇｅｎｅ（１９８５）３３：１０３−１０９）にサブクローニングした。ＤＮＡ配列決定の後、正しいＤＮＡ配列を有する鎖Ｖ領域をコードする遺伝子を含有するプラスミドをｐＵＣ−ＳＫ２Ｖ_Ｌ と命名した。プラスミドｐＵＣ−ＳＫ２Ｖ_Ｌ を含有する大腸菌はＥｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ ＤＨ５α（ｐＵＣ−ＳＫ２Ｖ_Ｌ ）として工業技術院生命工学工業技術研究所（茨城県つくば市東１丁目１番３号）に、平成５年４月２１日に、ＦＥＲＭ ＢＰ−４２７０としてブタペスト条約に基き国際寄託された。正しいＤＮＡ配列を有するＨ鎖Ｖ領域およびＬ鎖Ｖ領域のＨｉｎｄＩＩＩ −ＢａｍＨＩ断片を切出し、Ｈ鎖Ｖ領域については発現ベクターＨＥＦ−Ｖ_Ｈ −ｇγ１に導入してＨＥＦ−ＳＫ２ｈ−ｇγ１を得、Ｌ鎖Ｖ領域についてはＨＥＦ−Ｖ_Ｌ −ｇｋに導入してＨＥＦ−ＳＫ２ｋ−ｇｋを得た。
【００８３】
さらに、Ｈ鎖Ｖ領域に於いて、ほとんどの抗体のＨ鎖は、位置３０のセリンが保存されているが、マウスＳＫ２抗体Ｈ鎖においては位置３０がアスパラギンであることから、キメラＳＫ２抗体の抗原結合活性に対するこの変化の効果を検討するため、ＨＥＦ−ＳＫ２ｈ−ｇγ１を鋳型として後方プライマーＳＫ２Ｈ１Ｓと前方プライマーＳＫ２ＨＭ１Ｂ（配列番号：３２）及び前方プライマー６４ｈｃｈ−Ａと後方プライマー（ＳＫ２ＨＭ１）（配列番号：３３）の組合せを用いてＰＣＲ変異誘導法により、位置３０がアスパラギンからセリンに変るように設計した。
【００８４】
変異型Ｈ鎖Ｖ領域は、プライマーＳＫ２Ｈ１ＳとＳＫ２ＨＭ１Ｂ、又はプライマーＳＫ２ＨＭ１と６４ｈｃｈ−Ａを用い、鋳型ＤＮＡとしてプラスミドＨＥＦ−ＳＫ２ｈ−ｇγ１を用い、前記と同様にしてＰＣＲを行った。ＰＣＲ増幅の後、２つのＰＣＲ生成物を低融点アガロースゲルを用いて精製し、第２のＰＣＲのために用いた。
【００８５】
１μｇずつの２つの第一ＰＣＲ生成物及び５ｕのＡｍｐｌｉ Ｔａｑを含有する９８μｌのＰＣＲ混合物を９４℃にて２分間、５０℃にて２分間及び７２℃にて５分間インキュベートした後、１００ｐｍｏｌｅ ずつの外部プライマー（ＳＫ２Ｈ１Ｓ及び６４ｈｃｈ−Ａ）を加えた。ＰＣＲ混合物を５０μｌの鉱油で覆い、そして前記と同じ条件で第二ＰＣＲを行った。４１２ｂｐのＤＮＡ断片を精製し、ＨｉｎｄＩＩＩ 及びＢａｍＨＩで消化し、そして発現ベクターＨＥＦ−Ｖ_Ｈ −ｇγ１に挿入し、配列決定の後、正しい配列を含むプラスミドＨＥＦ−ＳＫ２ｈ−ＮＴＳを得た。
【００８６】
ＣＯＳ細胞へのトランスフェクション
キメラＳＫ２抗体の一過性発現を観察するため、前記発現ベクターをＣＯＳ細胞において試験した。ＨＥＦ−ＳＫ２ｋ−ｇｋと、ＨＥＦ−ＳＫ２ｈ−ｇγ１又はＨＥＦ−ＳＫ２ｈ−ＮＴＳのいずれかとを、Ｇｅｎｅ Ｐｕｌｓｅｒ装置（ＢｉｏＲａｄ社製）を用いてエレクトロポレーションによりＣＯＳ細胞に同時形質転換した。各ＤＮＡ（１０μｇ）を、ＰＢＳ中１×１０^７ 細胞／ｍｌの０．８ｍｌのアリコートに加え、１，９００Ｖ、２５μＦの容量にてパルスを与えた。
【００８７】
室温にて１０分間の回復期間の後、エレクトロポレーション処理された細胞を、ツニカマイシン（Ｓｉｇｍａ社製）を２μｇ／ｍｌ含有するか、又は含有しない１０％のγ−グロブリンフリーウシ胎児血清を含有するＤＭＥＭ培養液（ＧＩＢＣＯ社製）に加えた。７２時間のインキュベーションの後、培養上清を集め、遠心分離により細胞破片を除去し、５倍量の結合緩衝液で平衡化したプロテインＡアガロースカラム（Ａｆｆｉ−Ｇｅｌ Ｐｒｏｔｅｉｎ Ａ ＭＡＰＳＩＩキット、ＢｉｏＲａｄ）に適用した。カラムを１５倍量の結合緩衝液で洗浄し、次に５倍量の溶出緩衝液で溶出した。溶出液を濃縮し、そしてマイクロコンセントレーター（Ｃｅｎｔｒｉｃｏｎ １００，Ａｍｉｃｏｎ社製）を用いて緩衝液をＰＢＳに変えた。
【００８８】
ＥＬＩＳＡ
抗原結合測定のためのＥＬＩＳＡプレートを次の様にして調製した。９６穴プレートを、ＳＫ２が認識するエピトープとは異るヒトＩＬ−６のエピトープを認識するマウスモノクローナル抗体ＭＨ１６６（Ｍａｔｓｕｄａ，Ｔ．ら、Ｅｕｒ．Ｊ．Ｉｍｍｕｎｏｌ．１８，９５１−９５６，１９８８）でコートし、ブロッキングの後、組換えヒトＩＬ−６を添加した。キメラＳＫ２抗体のサンプルを順次希釈して、各穴に加え、次にアルカリホスファターゼ結合ヤギ抗−ヒトＩｇＧ抗体（Ｓｉｇｍａ社製）を加えた。インキュベーション及び洗浄の後、基質溶液を加え、次に４０５ｎｍでの吸光度を測定した。
【００８９】
結合阻害測定のため、ＥＬＩＳＡプレートを次の様にして調製した。９６穴プレートを、ヒトＩＬ−６レセプター（ＩＬ−６Ｒ）に対して特異的なマウスモノクローナル抗体ＭＴ−１８（Ｈｉｒａｔａ，Ｙ．ら、Ｊ．Ｉｍｍｕｎｏｌ．，１４３，２９００−２９０７，１９８９）によりコートし、ブロッキングの後、可溶性組換えヒトＩＬ−６Ｒ（ＳＲ３４４）（Ｙａｓｕｋａｗａ，Ｋ．ら、Ｊ．Ｂｉｏｃｈｅｍ．１０８，６７３−６７６，１９９０）を加えた。サンプルを順次希釈して、ビオチン化ＩＬ−６と共に各穴に加え、次いでアルカリホスファターゼ結合ストレプタビディンを加えた後、前記のようにして、４０５ｎｍにおける吸光度を測定した。
【００９０】
抗原結合測定の結果を図４に示し、結合阻害測定の結果を図５に示す。Ｈ鎖Ｖ領域の３０位がアスパラギンである。キメラ抗体およびＨ鎖Ｖ領域の３０位がセリンに変異しているキメラ抗体（ＮＴＳ）は、ツニカマイシンによる糖付加阻害の影響を受けることなく、同様の抗原結合性及び結合阻害活性を示した。従って、Ｈ鎖Ｖ領域の３０位のアミノ酸の変異及び糖鎖の有無は抗原結合性に何ら影響を与えないことが明らかになった。
【００９１】
ＳＤＳ／ＰＡＧＥ及びウエスタンブロットを用いたマウスＳＫ２抗体の糖鎖の分析
マウスＳＫ２抗体からＦａｂ断片およびＦｃ断片をＩｍｍｕｎｏ Ｐｕｒｅ Ｆａｂ Ｐｒｅｐａｒａｔｉｏｎ ｋｉｔ（Ｐｉｅｒｃｅ社製）を用いて調製し、次にＮ−グリカナーゼ（Ｇｅｎｚｙｍｅ社製）を用いて脱グリコシル化された断片を調製した。
【００９２】
これらの断片をＳＤＳ−ポリアクリルアミドゲル電気泳動にかけ、そして次にクマシーブリリアントブルー（Ｃｏｏｍａｓｓｉｅ Ｂｒｉｌｌｉａｎｔ Ｂｌｕｅ，ＢｉｏＲａｄ社製）により染色するか、又は電気泳動的にニトロセルロースフィルターに移行させた。Ｆｄ断片に付加されている糖を分析するため、フィルターをレクチンのパネル（Ｌｅｃｔｉｎ−Ｌｉｎｋ，Ｇｅｎｚｙｍｅ社製）にかけた。
【００９３】
図３に示すごとく、マウスＳＫ２抗体からのＦｄ断片はＳＤＳ／ＰＡＧＥにおいて約３４ｋｄ及び３３ｋｄのバンドを示し、この比率は約１：１であった。Ｎ−グリカナーゼにより脱グリコシル化されたＦｄ断片は約２９ｋｄの分子量を示し、これはＦｄ断片のアミノ酸組成から計算した値と一致した。また、３４ｋｄのＦｄ断片と３３ｋｄのＦｄ断片は種々のレクチンに対して異る反応性を示した。
【００９４】
すなわち３４ｋｄのＦｄはＤａｔｕｒａ Ｓｔｒａｍｏｎｉｕｍ Ａｇｇｌｕｔｉｎｉｎ（ＤＳＡ）＋、Ｒｉｃｉｎｕｓ Ｃｏｍｍｕｎｉｓ Ａｇｇｌｕｔｉｎｉｎ（ＲＣＡ）＋＋、Ｐｈａｓｅｏｌｕｓ Ｖｕｌｇａｒｉｓ Ｅｒｙｔｈｒｏｌｅｃｔｉｎ（ＰＨＡ−Ｅ）＋＋＋、Ｃｏｎｃａｎａｖａｌｉｎ Ａ（ＣｏｎＡ）−、Ｗｈｅａｔ Ｇｅｒｍ Ａｇｇｌｕｔｉｎｉｎ（ＷＧＡ）−であるのに対して、３３ｋｄのＦｄはＤＳＡ−，ＲＣＡ＋，ＰＨＡ−Ｅ＋＋＋，ＣｏｎＡ−，ＷＣＡ−であった。これらの結果、マウスＳＫ２抗体は、Ｈ鎖Ｖ領域に少なくとも２つのタイプのＮ型糖鎖を有することが明らかになった。
【００９５】
実施例５．再構成ヒトＳＫ２抗体の作製
再構成ヒトＳＫ２抗体Ｌ鎖Ｖ領域の作製
再構成ヒトＳＫ２抗体Ｌ鎖を、ＰＣＲ法によるＣＤＲ−グラフティングにより作製した。この方法を図６に模式的に示す。ヒト抗体ＲＥＩ由来のＦＲを有する再構成ヒト抗体ＳＫ２（バージョン「ａ」）の作製のために８個のＰＣＲプライマーを使用した。外部プライマーＡ（配列番号：３４）及びＨ（配列番号：３５）は、ｐＵＣベクターのＤＮＡ配列とハイブリダイズするように設計された。
【００９６】
ＣＤＲ−グラフティングプライマーＢ（配列番号：３６）、Ｃ（配列番号：３７）及びＤ（配列番号：３８）はセンスＤＮＡ配列を有し、そしてＣＤＲ−グラフティングプライマーＥ（配列番号：３９）、Ｆ（配列番号：４０）及びＧ（配列番号：４１）はアンチ−センスＤＮＡ配列を有しそしてそれぞれプライマーＢ，Ｃ及びＤの５′−末端のＤＮＡ配列に対する相補的ＤＮＡ配列（１５〜３５ｂｐ）を有する。
【００９７】
第一ＰＣＲ段階において４つの反応Ａ−Ｅ，Ｂ−Ｆ，Ｃ−Ｇ、及びＤ−Ｈを行い、そして各ＰＣＲ生成物を精製した。第一ＰＣＲからの４つのＰＣＲ生成物をそれら自体の相補性によりアッセンブリさせた（ＷＯ９２−１９７５９参照）。次に、外部プライマーＡ及びＨを加えて、再構成ヒトＳＫ２抗体Ｌ鎖Ｖ領域をコードする全長ＤＮＡを増幅した（第２ＰＣＲ）。前記ＰＣＲにおいては、ヒト抗体ＲＥＩからのＦＲに基く再構成ヒトＰＭ−１抗体Ｌ鎖Ｖ領域バージョン「ａ」をコードするプラスミドｐＵＣ−ＲＶＬ−ＰＭ１ａ−４（ＷＯ９２−１９７５９）を鋳型として用いた。
【００９８】
第一ＰＣＲ段階においては、１０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．３），５０ｍＭＫＣｌ，２５０μＭ ｄＮＴＰｓ，１．５ｍＭ ＭｇＣｌ_２ 、１００ｎｇの鋳型ＤＮＡ、１００ｐｍｏｌｅ の各プライマー及び５ｕのＡｍｐｌｉ Ｔａｑを含有する１００μｌのＰＣＲ混合物を用いた。各ＰＣＲチューブは５０μｌの鉱油で覆膜した。最初に９４℃で変性した後、９４℃にて１分間、５０℃又は５５℃にて１分間及び７２℃にて１分間の反応サイクルを行い、次に７２℃にて１０分間インキュベートした。
【００９９】
ＰＣＲ生成物Ａ−Ｅ（１８７ｂｐ），Ｂ−Ｆ（８７ｂｐ），Ｃ−Ｇ（１５２ｂｐ）及びＤ−Ｈ（６５ｂｐ）を１．５％低融点アガロースゲルを用いて精製し、第二ＰＣＲでアッセンブリした。第二ＰＣＲにおいては、１μｇの各第一ＰＣＲの生成物、及び５ｕのＡｍｐｌｉ Ｔａｑを含有する９８μｌのＰＣＲ混合物を、９４℃にて２分間、５０℃にて２分間及び７２℃にて２分間で２サイクルインキュベートし、そして次に１００ｐｍｏｌｅ の各外部プライマー（Ａ及びＨ）を加えた。ＰＣＲチューブを５０μｌの鉱油で覆い、そして前記と同一の条件で３０サイクルのＰＣＲを行った。
【０１００】
第二ＰＣＲにより生じた３９９ｂｐのＤＮＡ断片を１．５％低融点アガロースゲルで精製し、ＢａｍＨＩ及びＨｉｎｄＩＩＩ で消化し、得られたＤＮＡ断片をＨＥＦ発現ベクターＨＥＦ−Ｖ_Ｌ −ｇｋにクローニングした。ＤＮＡ配列決定の後、再構成ヒトＳＫ２抗体Ｌ鎖Ｖ領域のバージョン「ａ」の正しいアミノ酸配列をコードするＤＮＡ断片を含むプラスミドをＨＥＦ−ＲＶＬ−ＳＫ２ａと命名した。本プラスミドＨＥＦ−ＲＶＬ−ＳＫ２ａに含まれるＬ鎖Ｖ領域のアミノ酸配列および塩基配列を配列番号：５２に示す。
【０１０１】
再構成ヒトＳＫ２抗体Ｌ鎖Ｖ領域のバージョン「ｂ」を、ＰＣＲを用いる変異誘発によって作製した。変異原プライマーＦＴＹ−１（配列番号：４２）及びＦＴＹ−２（配列番号：４３）は、７１位のフェニルアラニンがチロシンに変異するように設計した。プラスミドＨＥＦ−ＲＶ１−ＳＫ２ａを鋳型ＤＮＡとして用いた。最終ＰＣＲ生成物を精製し、ＢａｍＨＩ及びＨｉｎｄＩＩＩ で消化し、得られたＤＮＡ断片をＨＥＦ−発現ベクターＨＥＦ−Ｖ_Ｌ −ｇｋにクローニングしてプラスミドＨＥＦ−ＲＶＬ−ＳＫ２ｂを得た。本プラスミドＨＥＦ−ＲＶＬ−ＳＫ２ｂに含まれるＬ鎖Ｖ領域のアミノ酸配列および塩基配列を配列番号：５３に示す。
【０１０２】
再構成ヒトＳＫ２抗体Ｈ鎖Ｖ領域の作製
再構成ヒトＳＫ２抗体Ｈ鎖Ｖ領域をコードするＤＮＡを次の様にして設計した。ヒト抗体ＤＡＷのＦＲをコードするＤＮＡ配列をＶ領域（Ｋａｂａｔ，Ｅ．Ａ．ら、ＵＳ Ｄｅｐｔ Ｈｅａｌｔｈ ａｎｄ Ｈｕｍａｎ Ｓｅｒｖｉｃｅｓ，ＵＳ Ｇｏｖｅｒｎｍｅｎｔ Ｐｒｉｎｔｉｎｇ Ｏｆｆｉｃｅｓ，１９９１）の「ｃｏｄｏｎ ｕｓａｇｅ」に基いて設計し、そしてマウスＳＫ２抗体Ｈ鎖Ｖ領域のＣＤＲをコードするＤＮＡ配列とつなげることにより、再構成ヒトＳＫ２抗体Ｈ鎖Ｖ領域のバージョン「ａ」をコードする全長ＤＮＡを設計した。
【０１０３】
次に、このＤＮＡ配列のそれぞれ５′−側及び３′−側にＨｉｎｄＩＩＩ 認識部位／ＫＯＺＡＫコンセンサス配列及びＢａｍＨＩ認識部位／スプライスドナー配列を付加して、ＨＥＦ発現ベクターに挿入できるようにした。
こうして設計したＤＮＡ配列を６個のオリゴヌクレオチドに分け、そして次に、これらのオリゴヌクレオチドのアセンブリーを妨害する可能性のあるオリゴヌクレオチド中の二次構造についてコンピューター解析した。
【０１０４】
６個のオリゴヌクレオチド配列を配列番号：４４〜４９に示す。これらのオリゴヌクレオチドは９３〜９６塩基の長さを有し、２０〜２４ｂｐのオーバラップ領域を有する。オリゴヌクレオチドの内の３個、すなわち、ＨＰ１（配列番号：４４）、ＨＰ３（配列番号：４６）及びＨＰ５（配列番号：４８）はセンスＤＮＡ配列を有し、そして他の３個、すなわち、ＨＰ２（配列番号：４５）、ＨＰ４（配列番号：４７）及びＨＰ６（配列番号：４９）はアンチセンスＤＮＡ配列を有する。これら６個のオリゴヌクレオチドのＰＣＲ法によるアセンブリーの方法を図７に示す。
【０１０５】
２ｐｍｏｌｅ ずつの６種のオリゴヌクレオチド及び５ｕのＡｍｐｌｉ Ｔａｑを含有する９８μｌのＰＣＲ混合物を、９４℃にて２分間の最初の変性の後、９４℃にて１分間、５０℃にて１分間及び７２℃にて１分間のから成る３サイクルのインキュベーションを行い、次に７２℃にて１０分間インキュベートした。５０μｍｏｌｅずつのＨＰ１及びＨＰ６を外部プライマーとして添加した後、ＰＣＲチューブを５０μｌの鉱油で覆い、そして９４℃にて１分間の最初の変性の後、９４℃にて１分間、５０℃にて１分間及び７２℃にて１分間の３０サイクルを行い、そして次に７２℃にて１０分間インキュベートした。
【０１０６】
４４８ｂｐのＤＮＡ断片を１．５％低融点アガロースゲルを用いて精製し、ＨｉｎｄＩＩＩ 及びＢａｍＨＩにより消化し、そして次にＨＥＦ発現ベクターＨＥＦ−Ｖ_Ｈ −ｇγ１にクローニングした。ＤＮＡ配列決定の後、正しいＨ鎖Ｖ領域のアミノ酸配列をコードするＤＮＡ断片を含むプラスミドをＨＥＦ−ＲＶＨ−ＳＫ２ａと命名した。本プラスミドＨＥＦ−ＲＶＨ−ＳＫ２ａに含まれるＨ鎖Ｖ領域のアミノ酸配列および塩基配列を配列番号：５４に示す。
【０１０７】
バージョン「ｂ」を、ＰＣＲ法による変異誘発を用いて作製した。変異原プライマーＲＳＫ２−ＳＴＮＡ（配列番号：５０）及びＲＳＫ−ＳＴＮＢ（配列番号：５１）を、３０位のセリンがアスパラギンに変る様に設計した。プラスミドＲＶＨ−ＳＫ２ａを鋳型ＤＮＡとして使用した。最終ＰＣＲ生成物を精製し、ＢａｍＨＩ及びＨｉｎｄＩＩＩ により消化し、そして次にＨＥＦ発現ベクターＨＥＦ−Ｖ_Ｈ −ｇγ１にクローニングし、プラスミドＨＥＦ−ＲＶＨ−ＳＫ２ｂを得た。本プラスミドＨＥＦ−ＲＶＨ−ＳＫ２ｂに含まれるＨ鎖Ｖ領域のアミノ酸配列および塩基配列を配列番号：５５に示す。
【０１０８】
再構成ヒトＳＫ２抗体の各鎖を評価するため、まず、再構成ヒトＳＫ２抗体Ｌ鎖のための２種類の発現ベクターＨＥＦ−ＲＶＬ−ＳＫ２ａ（バージョン「ａ」）及びＨＥＦ−ＲＶＬ−ＳＫ２ｂ（バージョン「ｂ」）のいずれかと、キメラＳＫ２抗体Ｈ鎖のための発現ベクターＨＥＦ−ＳＫ２ｈ−ｇγ１とによりＣＯＳ細胞を前記のようにして同時トランスフェクションし、前記のようにして抗体生成物を回収した後、前記のようにして抗原結合測定にかけた。
【０１０９】
この結果を図８に示す。図８に示すように、陽性対照としてのキメラ抗体（ＣｈＬ／ＣｈＨ）と、再構成Ｌ鎖バージョン「ａ」とキメラＨ鎖とから成る抗体（ＲＶＬａ／ＣｈＨ）、と再構成Ｌ鎖バージョン「ｂ」とキメラＨ鎖とから成る抗体（ＲＶＬｂ／ＣｈＨ）との間には抗原結合性に差がなく、７１位におけるチロシン残基のフェニルアラニン残基への変化は抗原結合性に影響を与えないことが示された。
【０１１０】
次に、再構成ヒト化ＳＫ２抗体Ｌ鎖の２つのバージョンと再構成ヒト化ＳＫ２抗体Ｈ鎖の２つのバージョンとの組合せを評価するため、ＨＥＦ−ＲＶＬ−ＳＫ２ａとＨＥＦ−ＲＶＨ−ＳＫ２ａ、ＨＥＦ−ＲＶＬ−ＳＫ２ａとＨＥＦ−ＲＶＨ−ＳＫ２ｂ、ＨＥＦ−ＲＶＬ−ＳＫ２ｂとＨＥＦ−ＲＶＨ−ＳＫ２ａ、又はＨＥＦ−ＲＶＬ−ＳＫ２ｂとＨＥＦ−ＲＶＨ−ＳＫ２ｂとによりＣＯＳ細胞を同時トランスフェクションし、前記のようにして得られた抗体について抗原結合測定を行った。その結果を図９に示す。図９から明らかな通り、再構成ヒトＨ鎖と再構成ヒトＶ鎖とのすべての組合せが、キメラＳＫ２抗体に匹敵する良好な抗原結合性を示した。
【０１１１】
なお、前記プラスミドＨＥＦ−ＲＶ_Ｌ −ＳＫ２ｂを含有する大腸菌はＥｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ ＤＨ５α（ＨＥＦ−ＲＶ_Ｌ ＳＫ２ｂ）、およびプラスミドＨＥＦ−ＲＶ_Ｈ −ＳＫ２ａを含有する大腸菌はＥｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ ＤＨ５α（ＨＥＦ−ＲＶ_Ｈ ＳＫ２ａ）として工業技術院生命工学工業技術研究所（茨城県つくば市東１丁目１番３号）に、平成５年４月２１日に、各々ＦＥＲＭ ＢＰ−４２６７、およびＦＥＲＭ ＢＰ−４２６８としてブダペスト条約に基き国際寄託された。
【０１１２】
これに対して、図１０に示すように、結合阻害（ＩＬ６とＩＬ−６Ｒと結合を阻害する抗体の能力）測定においては、再構成ヒトＳＫ２抗体Ｌ鎖バージョン「ｂ」（ＲｖＬｂ）と再構成ヒトＳＫ２抗体Ｈ鎖バージョン「ａ」（ＲｖＨａ）との組合せのみが、マウスモノクローナル抗体ＳＫ２及びキメラＳＫ２抗体に匹敵する結合阻害活性を示し、この組合せがヒト抗体における機能的抗原結合部位を形成することが示唆された。
【０１１３】
他方、再構成ヒトＳＫ２抗体Ｌ鎖バージョン「ａ」（ＲｖＬａ）と再構成ヒトＳＫ２抗体Ｈ鎖バージョン「ａ」（ＲｖＨａ）との組合せ、及びＲｖＬａとＲｖＨｂとの組合せは低い結合阻害活性を示し、Ｌ鎖中の７１位にフェニルアラニンを有する抗体は良好な抗原結合活性を示すが機能的抗原結合部位を再形成することが示唆された。
これらのことからヒト抗体Ｌ鎖のＣＤＲ１領域に於けるループ構造には、７１位のチロシンは機能的な抗原結合部位を再構成するのに必須であると言える。
【０１１４】
参考例１． ハイブリドーマＳＫ２の作製
抗−ヒトＩＬ−６モノクローナル抗体を産生するハイブリドーマはヒトＩＬ−６で免役したＢＡＬＢ／ｃマウスの脾臓細胞とマウス骨髄腫細胞株Ｐ３Ｕ１をポリエチレングリコールを用いた常法により融合して作製した。ＩＬ−６とＩＬ−６Ｒの結合を阻害する活性を指標としたスクリーニングを行い、結合阻害活性を有する１７種のクローンを得た。これらのクローンをマウスの腹腔内に移植し、得られた腹水をプロテインＡアガロースカラムにかけ、精製マウスモノクローナル抗体を得た。
【０１１５】
参考例２． マウスモノクローナル抗体ＳＫ２のタイピング
ハイブリドーマＳＫ２から産生されるマウスモノクローナル抗体ＳＫ２のＬ鎖およびＨ鎖のタイプを調べるために、マウスモノクローナル抗体アイソタイピングキット（アマシャムインターナショナルｐｌｃ社製）を用いてタイピングを行った。その結果、ＳＫ２抗体はκ型軽鎖およびγ１型重鎖を有することが明らかになった。
【０１１６】
【配列表】

【０１１７】

【０１１８】

【０１１９】

【０１２０】

【０１２１】

【０１２２】

【０１２３】

【０１２４】

【０１２５】

【０１２６】

【０１２７】

【０１２８】

【０１２９】

【０１３０】

【０１３１】

【０１３２】

【０１３３】

【０１３４】

【０１３５】

【０１３６】

【０１３７】

【０１３８】

【０１３９】

【０１４０】

【０１４１】
配列番号：２６
配列の長さ：３７９
配列の型：核酸
鎖の数：二本鎖
トポロジー：直鎖状
配列の種類：ｃＤＮＡ
起源
生物名：マウス
直接の起源
クローン：ｐｓｋ２−ｋ２
特徴： １．．６０ ｓｉｇ ｐｅｐｔｉｄｅ
６１．．２７９ ｍａｔ ｐｅｐｔｉｄｅ
配列
【０１４３】

【０１４４】

【０１４５】

【０１４６】

【０１４７】

【０１４８】

【０１４９】

【０１５０】

【０１５１】

【０１５２】

【０１５３】

【０１５４】

【０１５５】

【０１５６】

【０１５７】

【０１５８】

【０１５９】

【０１６０】

【０１６１】

【０１６２】

【０１６３】

【０１６４】

【０１６５】

【０１６６】

【０１６７】

【０１６８】

【０１６９】

【０１７０】

【０１７１】

【０１７２】

【０１７３】

【０１７５】

【０１７６】

【０１７７】

【０１７９】

【０１８０】

【図面の簡単な説明】
【図１】図１は、それぞれＬ鎖及びＨ鎖の発現のために有用な、ヒトＥＦＩ−αプロモーター／エンハンサーを含んで成る発現プラスミドＨＥＦ−Ｖ_Ｌ −ｇｋ及びＨＥＦ−Ｖ_Ｈ −ｇγ１を示す。
【図２】図２は、本発明の抗体ペプチドの発現のために有用な、ヒトサイトメガロウイルス（ＨＣＭＶ）プロモーター／エンハンサー系を含んで成る発現ベクターを示す。
【図３】図３は、ＳＫ２抗体がグリコシル化されていることを示す電気泳動図である。
【図４】図４は、ヒトＩＬ−６に結合する本発明のキメラ抗体の能力の確認のためのＥＬＩＳＡの結果を示すグラフである。
【図５】図５は、ヒトＩＬ−６へのＩＬ−６の結合を阻害する本発明のキメラ抗体の能力の確認のためのＥＬＩＳＡの結果を示すグラフである。
【図６】図６は、再構成ヒトＳＫ２抗体Ｌ鎖Ｖ領域の第一バージョン（バージョン「ａ」）の作製のダイヤグラムである。
【図７】図７は、再構成ヒトＳＫ２抗体Ｈ鎖Ｖ領域の第一バージョン（バージョン「ａ」）の作製のダイヤグラムである。
【図８】図８は、ヒトＩＬ−６への結合についての、再構成ヒトＬ鎖＋キメラＨ鎖から成る抗体の能力を示すグラフである。
【図９】図９は、ヒトＩＬ−６への結合についての、本発明の４種類の再構成ヒトＳＫ２抗体の能力をキメラ抗体のそれと比較して示すグラフである。
【図１０】図１０は、ＩＬ−６ＲへのＩＬ−６の結合を阻害する本発明の４種類の再構成ヒト化ＳＫ２抗体の能力を、ＳＫ２キメラ抗体及びマウスモノクローナル抗体ＳＫ２のそれと比較して示すグラフである。[0001]
[Industrial application fields]
The present invention relates to a human / mouse chimeric antibody comprising a variable region (V region) of mouse monoclonal antibody SK2 against human interleukin-6 (IL-6) and a constant region (C region) of human antibody, human light chain (L A reshaped human antibody in which the complementarity determining region (CDR) of the chain) V region and the human heavy chain (H chain) V region is replaced by the CDR of the mouse monoclonal antibody SK2 against human IL-6 It relates to the constituting L chain or H chain.
[0002]
The present invention further provides DNA encoding the above antibody, particularly the V region thereof. The invention further relates to a vector comprising said DNA, in particular an expression vector, and a host transformed with said vector. The present invention further provides a method for producing a chimeric antibody against human IL-6 and a method for producing a reshaped human antibody against human IL-6.
[0003]
[Prior art]
Interleukin-6 (IL-6) is a multifunctional cytokine produced by a series of cells. It regulates immune responses, acute phase responses and hematopoiesis and plays a central role in host defense mechanisms (Kishimoto et al., Blood,74, 1-10, 1989). This acts on a wide range of tissues and exhibits growth-inducing effects, growth-inhibiting effects, and differentiation-inducing effects depending on the nature of the target cells.
[0004]
It has been suggested that abnormal expression of the IL-6 gene is involved in the pathogenesis of various diseases, particularly autoimmune diseases, mesangial cell proliferative glomerulonephritis, and plasmacytoma / myeloma (Hirano et al., Immunol. Today). ,11443-449, 1990; Clein, B .; Et al., Eur. Cytokine Net.1, 193-201, 1990). Therefore, antibodies that inhibit IL-6 function are expected to be useful as therapeutic agents in human patients.
[0005]
In fact, in clinical studies using mouse monoclonal antibodies against human IL-6 to treat end-stage patients with plasmacytomas, blocking the proliferation of plasmacytoma cells, and circulating M protein, serum calcium, serum IgG and C -A decrease in reactive protein was shown (Klein, B. et al., Blood,78, 1198-1204, 1991; Klein, B et al., Res. Immunol. ,143774-776, 1992).
[0006]
Murine monoclonal antibodies are highly immunogenic (sometimes referred to as “antigenic”) in humans, and for this reason, their therapeutic value in humans is limited. Furthermore, murine antibodies cannot be administered frequently without interfering with the expected effects and without eliciting an immune response that poses a risk of an adverse allergic response in the patient.
[0007]
In order to solve these problems, a method for producing a humanized antibody has been developed. Mouse antibodies can be humanized in two ways. A simpler method is to make a chimeric antibody in which the variable region is derived from the original mouse monoclonal antibody and the constant region is derived from a suitable human antibody. The resulting chimeric antibody contains the complete variable region of the original mouse antibody and can be expected to bind to the antigen with the same specificity as the original mouse antibody.
[0008]
In addition, chimeric antibodies are expected to have a substantially reduced proportion of protein sequences derived from non-humans and are therefore expected to be less immunogenic than the original mouse antibodies. Chimeric antibodies bind well to antigens and are less immunogenic, but an immune response against the murine variable region may still occur (Lo Buglio et al., Proc. Natl. Acad. Sci. USA,864220-4224, 1989).
[0009]
The second method for humanizing mouse antibodies is more complex, but significantly reduces the potential immunogenicity of mouse antibodies. In this method, a complementarity determining region (CDR) from a variable region of a mouse antibody is transplanted into a human antibody variable region to create a “reshaped” human antibody variable region.
[0010]
These reshaped human variable regions are then linked to human antibody constant regions. The part derived from the non-human protein sequence of the finally reconstituted human-type antibody is only CDR and a part of framework (FR). CDRs are composed of hypervariable protein sequences. They do not show species specific sequences. For these reasons, reconstituted human antibodies carrying mouse CDRs should no longer be more immunogenic than natural human antibodies containing human CDRs.
[0011]
For reconstituted human antibodies, see Riechmann, L. et al. Et al., Nature,332, 323-327, 1988; Verhoey, M .; Science,239, 1534-1536, 1988; Kettleborough, C .; A. Et al., Protein Enng.4, 773-783, 1991; Maeda, H .; Et al., Human Antibodies and Hybridoma,2, 124-134, 1991; Gorman, S .; D. Et al., Proc. Natl. Acad. Sci. USA,88, 4181-4185, 1991; Tempest, P .; R. Et al., Bio / Technology,9, 266-271, 1991; S. Et al., Proc. Natl. Acad. Sci. USA,88, 2869-2873, 1991; Carter, P .; Et al., Proc. Natl. Acad. Sci. USA,89, 4285-4289, 1992; M.M. S. Et al. Immunol.148, 1149-1154, 1992; and Sato, K .; Et al., Cancer Res.53, 1-6, 1993.
[0012]
[Problems to be solved by the invention]
As noted above, reshaped human antibodies are expected to be useful for therapeutic purposes, but reshaped human antibodies against human IL-6 are not known. Furthermore, there are no methods for producing reshaped human antibodies that can be universally applied to any particular antibody. Therefore, various devices are required to produce a reshaped human antibody that is sufficiently active against a specific antigen (see, for example, Sato, K. et al., Cancer Res.53851-856 (1993)).
[0013]
The present inventors have found that the mouse monoclonal antibody SK2 against human IL-6 strongly inhibits the function of IL-6 in in vivo animal experiments and can therefore be expected as a therapeutic agent for IL-6 related diseases (Saito, H Et al., 21st Annual Meeting of the Immunological Society of Japan, Academic Meeting Record,21, 116, 1991). Accordingly, the present invention seeks to provide antibodies with low immunogenicity against human IL-6.
[0014]
[Means for Solving the Problems]
Accordingly, the present invention relates to a reshaped human antibody of mouse monoclonal antibody SK2 against human IL-6. The present invention also provides a human / mouse chimeric antibody useful in the process of producing the reshaped human antibody. The invention further relates to genetic engineering methods for the production of reshaped human antibodies as well as chimeric antibodies of mouse monoclonal antibody SK2.
[0015]
Specifically, the present invention provides:
(1) an L chain comprising a human antibody L chain C region and an L chain V region of a mouse monoclonal antibody SK2 against human IL-6; and
(2) the H chain comprising the human antibody H chain C region and the H chain V region of the mouse monoclonal antibody SK2 against human IL-6;
A chimeric antibody to human IL-6, comprising:
(1) framework region (FR) of human antibody L chain V region, and
(2) CDR of L chain V region of mouse monoclonal antibody SK2 against human IL-6,
A reshaped human L chain V region comprising:
(1) FR of human antibody H chain V region, and
(2) CDR of H chain V region of mouse monoclonal antibody SK2 against human IL-6,
The mouse monoclonal antibody SK2 against the human IL-6 composed of the reshaped human H chain V region comprising
The present invention also relates to L-chain or H-chain polypeptides constituting the various antibodies, and DNAs encoding them.
Furthermore, the present invention provides an expression vector comprising the above DNA and
The present invention relates to a host transformed with these expression vectors.
The present invention further relates to a method for producing a chimeric antibody against human IL-6 and a method for producing a reshaped human antibody against human IL-6.
[0016]
[Specific explanation]
Cloning of DNA encoding mouse antibody V region
In order to clone DNA encoding the V region of a mouse monoclonal antibody against human IL-6, total RNA was prepared from a mouse monoclonal antibody-producing cell, and then converted into a single-stranded cDNA by a known method. It can be obtained by amplifying the target DNA using a reaction (PCR) method. As a source of this total RNA, it is necessary to produce a hybridoma that produces a monoclonal antibody against human IL-6. An example of such a hybridoma is SK2. A method for producing hybridoma SK2 is described later in Reference Example 1.
[0017]
(1) Collection of total RNA
In the present invention, in order to collect total RNA, hybridoma cells were disrupted, treated with guanidine thiocyanate, and then subjected to cesium chloride density gradient centrifugation (chirgwin et al., Biochemistry,18, 5294, 1979), which are already used for cloning genes of other proteins, for example, in the presence of a ribonuclease inhibitor such as a vanadium complex, are treated with a surfactant and treated with phenol (Berger, S. L., et al. Et al. Biochemistry,18, 5143, 1979) can be used.
[0018]
(2) Collection of single-stranded cDNA
To obtain single-stranded DNA from the total RNA obtained as described above, treat with reverse transcriptase using total RNA as a template and oligo (dT) complementary to the polyA strand at the 3 'end as a primer. To synthesize single-stranded DNA (cDNA) complementary to total RNA (Larrik, JW et al., Bio / Technology,7, 934, 1989). At that time, a random primer may be used.
[0019]
(3) Amplification of mouse antibody V region by polymerase chain reaction (PCR) method
Next, the mouse antibody V region is specifically amplified from the cDNA using the polymerase chain reaction (PCR) method. For amplification of the mouse antibody V region, Jones, S .; T.A. Et al. Bio / Technology,9, 88-89, 1991 can be used. In determining the primers used for the cloning of the mouse monoclonal antibody SK2 produced by the hybridoma SK2, it is necessary to determine the type of both chains by typing H and L chains.
[0020]
As a result of SK2 antibody typing using a mouse monoclonal antibody isotyping kit (Amersham International plc), it was revealed that the SK2 antibody has a κ-type L chain and a Cγ1-type H chain. The SK2 typing will be described later in Reference Example 2.
Next, in order to amplify the kappa (κ) type L chain V region of the mouse monoclonal antibody using the polymerase chain reaction (PCR) method, 11 kinds of oligonucleotide primers shown in SEQ ID NOs: 1 to 11 (Mouse Kappa Variable; MKV) and the oligonucleotide primer shown in SEQ ID NO: 12 (Mouse Kappa Constant; MKC) are used as a 5'-end primer and a 3'-end primer, respectively.
[0021]
The MKV primer hybridizes with a DNA sequence encoding a mouse kappa type L chain leader sequence, and the MKC primer hybridizes with a DNA sequence encoding a mouse kappa type L chain C region.
For amplification of the H chain V region of the mouse monoclonal antibody, 12 kinds of oligonucleotide primers (Mouse Heavy Variable; MHV) shown in SEQ ID NOs: 13 to 24 and an oligonucleotide primer (Mouse Heavy Constant; MHC) shown in SEQ ID NO: 25 ) Are used as 5'-end primer and 3'-end primer, respectively.
[0022]
In this example, the 5'-end primer contains the sequence GTCGAC providing a restriction enzyme SalI cleavage site near its 5'-end, and the 3'-end primer is located near its 5'-end with restriction enzyme XmaI. Those containing the nucleotide sequence CCCGGG providing the cleavage site are used. These restriction enzyme cleavage sites may be other restriction enzyme cleavage sites as long as they are used for subcloning a DNA fragment of interest encoding a variable region into a cloning vector.
[0023]
Next, in order to obtain a DNA fragment encoding the target variable region of the mouse monoclonal antibody, the amplification product was cleaved with restriction enzymes SalI and XmaI, and then a low melting point agarose or column [PCR product purification kit (QIAGEN etc. ), DNA purification kit (GENECLEAN II, etc.)] and the like. On the other hand, a plasmid containing a DNA fragment encoding a variable region of interest of a mouse monoclonal antibody by cleaving a suitable cloning vector such as plasmid pUC19 with the same restriction enzymes SalI and XmaI and ligating the DNA fragment to pUC19. Get.
[0024]
The cloned DNA can be sequenced using any conventional method, for example, Sequence Version 2.0 (manufactured by United States Biochemical Corporation).
The cloning and sequencing of the DNA of interest is specifically described in Examples 1 and 2.
[0025]
Complementarity determining region (CDR)
The V regions of each pair of L and H chains form an antigen binding site. The variable regions on the L and H chains are linked by four common and relatively conserved frameworks and three hypervariable or complementarity determining regions (CDRs) (Kabat, EA; Et al., “Sequences of Proteins of Immunological Interest”, US Dept. Health and Human Services 1983).
[0026]
Many parts of the four framework regions (FR) have a β-sheet structure, so that the three CDRs form a loop. The CDR may in some cases form part of the β-sheet structure. The three CDRs are held sterically very close to each other by the FR and contribute to the formation of an antigen binding site together with the three CDRs of the paired region.
[0027]
These CDR regions can be determined by comparing the amino acid sequence of the V region of the obtained antibody with the known amino acid sequence of the V region of a known antibody. A. Can be found from the empirical rule of “Sequences of Proteins of Immunological Interest”, which will be described in detail in Example 3.
[0028]
Production of chimeric antibody
Prior to designing the reshaped human V region of antibodies against human IL-6, it is necessary to make sure that the CDRs used actually form the antigen binding region. For this purpose, a chimeric antibody was produced. Furthermore, the amino acid sequence of the mouse anti-human IL-6 antibody deduced from the nucleotide sequence of the cloned DNA of the monoclonal antibody SK2 described in Example 1 was compared with the V regions of known mouse and human antibodies.
[0029]
Once the DNA fragments encoding the mouse L chain and H chain V regions of the monoclonal antibody SK2 have been cloned, these mouse V regions are expressed linked to DNA encoding the human antibody constant region to express chimeric anti-human IL- 6 antibodies are obtained.
The basic method for making a chimeric antibody is to link the mouse leader sequence and V region sequence present in the cloned cDNA to the sequence encoding the human antibody C region already present in the mammalian cell expression vector. Comprising that.
[0030]
The human antibody C region may be any human L chain C region and human H chain C region, and examples thereof include human L chain Cκ or H chain Cγ1 and Cγ4.
For the production of chimeric antibodies, two types of expression vectors, ie expression comprising DNA encoding mouse L chain V region and human L chain C region under the control of an expression control region such as an enhancer / promoter system. An expression vector comprising DNA encoding a mouse H chain V region and a human H chain C region under an expression control region such as an enhancer / promoter system is prepared. Next, a host cell such as a mammalian cell is co-transformed with these expression vectors, and the transformed cell is cultured in vitro or in vivo to produce a chimeric antibody (eg, WO 91-16928).
[0031]
Alternatively, DNA encoding mouse L chain V region and human L chain C region and DNA encoding mouse H chain V region and human H chain C region are introduced into a single expression vector, and the vector is used to host Cells are transformed and the transformed host is then cultured in-vivo or in-vitro to produce the desired chimeric antibody.
The production of chimeric antibodies is described in Example 4.
[0032]
A cDNA encoding the mouse SK2κ type L chain leader region and V region is subcloned using the PCR method and ligated to an expression vector containing DNA encoding the human genomic L chain Cκ chain region. The cDNA encoding the mouse SK2 antibody γ1-type H chain leader and V region is subcloned using the PCR method and ligated to an expression vector containing genomic DNA encoding the human γ-1C region.
[0033]
Using specially designed PCR primers, cDNAs encoding the V region of mouse SK2 are introduced into their expression vectors by introducing appropriate base sequences at their 5'- and 3'-ends. And so that they function properly in the expression vector (for example, the present invention is devised to increase transcription efficiency by introducing a Kozak sequence). Next, the V region of mouse SK2 obtained by PCR amplification using these primers was inserted into a HEF expression vector (FIG. 1) already containing the desired human C region. These vectors are suitable for transient or stable expression of genetically engineered antibodies in various mammalian cell lines.
[0034]
In addition to creating a chimeric SK2 antibody having the same V region as that present in the mouse SK2 antibody, a second version of the chimeric SK2 antibody (NTS) was created. In the chimeric SK2 antibody NTS, the amino acid at position 30 in the H chain V region is changed from asparagine to serine.
[0035]
Mouse heavy chain subgroup I (Fischmann, TO et al., J. Biol. Chem.266, 12915, 1991), the most typical amino acid at position 30 is serine (Kabat, EA, et al., US Dept Health and Human Services US Government Printing Offices, 1991).
[0036]
To assess the importance of having an atypical amino acid asparagine at position 30 in the H chain V region of the mouse SK2 antibody, position 30 was changed to serine, a typical amino acid. This change is based on the PCR-mutagenesis method (M. Kamman et al., Nucl. Acids Res.15, 5404, 1989) by making the necessary changes in the DNA sequence encoding the H chain V region. The chimeric SK2 antibody has a glycosylation site at position 30 of the H chain V region, similar to the original SK2 antibody, whereas the mutant chimeric SK2 antibody NTS has no glycosylation site.
[0037]
The chimeric SK2 antibody showed activity to bind to human IL-6. The chimeric SK2 antibody NTS also binds to human IL-6. Thus, it was shown that the correct mouse V region was cloned and sequenced. It was also shown that glycosylation of the SK2 antibody H chain V region is not necessary for the binding activity of the SK2 antibody.
[0038]
Construction of reconstituted human antibodies
In order to produce a reconstructed human antibody in which the CDR of the mouse monoclonal antibody is transplanted to the human antibody, it is desirable that high homology exists between the FR of the mouse monoclonal antibody and the FR of the human antibody. Therefore, the V regions of the L and H chains of the mouse SK2 antibody were compared with the V regions of all known antibodies whose structures were elucidated using the Protein Data Bank. The results are summarized in Table 1.
[0039]
[Table 1]

[0040]
The L chain V region of mouse SK2 is most similar to the consensus sequence of human L chain V region subgroup I (HSGI), with 60.3% homology. The H chain V region of mouse SK2 is most similar to the consensus sequence of human H chain V region subgroup II (HSGII), with 59.2% homology.
[0041]
From the comparison with the V region in the human antibody, it is possible to select the FR of the human V region that is the basis for the design of the reshaped human SK2 antibody V region. Human L chain V regions belonging to subgroup I (HSGI) are used for the design of reshaped human SK2 antibody L chain V regions, and subgroups for the design of reshaped human SK2 antibody H chain V regions It is better to use the human antibody H chain V region belonging to II (HSGII), and it would be best to select those with the highest homology within the subgroup (Kabat, EA et al., US Dep. Health and Human Services, US Government Printing Offices, 1991).
[0042]
Design of reshaped human SK2 antibody V region
The first step in designing the reshaped human SK2 antibody V region was to select the human antibody V region that was the basis for the design. The mouse SK2 antibody L chain V region was most similar to the human kappa type antibody L chain V region belonging to subgroup I (Table 1). The L chain V region of the mouse SK2 antibody shows 63% similarity to the human L chain V region REI, which is a member of the subgroup I of the human L chain V region, in comparison with the known human antibody L chain V region. It was. Therefore, REI FR was used as a starting material for the generation of reshaped human SK2 antibody L chain V region.
[0043]
Two versions of the reshaped human SK2 antibody L chain V region were designed. In the first version (version “a”), human FRs are based on the REI-based FRs (Riechmann, L. et al., Nature) present in reconstituted human CAMPATH-1H antibodies.332, 323-327, 1988) (version “a” of the L chain V region of reshaped human PM-1 as described in WO 92-19759) and the mouse CDR is the CDR in the L chain V region of mouse SK2. It was the same. The second version (version “b”) differs from version “a” by only one amino acid at position 71 in human FR3.
[0044]
That is, position 71 is phenylalanine in the modified REI FR used in the design of version “a”, whereas in version “b”, phenylalanine at position 71 is found in the mouse SK2 antibody L chain V region. It is changed to tyrosine. Table 2 shows the amino acid sequences of two versions of the L chain V region of the mouse SK2 antibody, the FR of REI, and the L chain V region of the reconstituted SK2 antibody.
[0045]
[Table 2]

Note: The five underlined amino acids in the FR of REI are the amino acid sequence of human REI (Palm, W. et al., Hoppe-Seyler's, Z. Physiol. Chem.,356, 167-191, 1975).
[0046]
The FRs in the H chain V region of the mouse SK2 antibody are most similar to the human H chain V region belonging to subgroup II (Table 1). In comparison with the known human antibody H chain V region, the H chain V region of the mouse SK2 antibody is a human H chain V region DAW (Press, E.M. Et al., Biochem.117, 641, 1970) (60.8%). Furthermore, the size of the CDR was also very similar between the mouse SK2 antibody and the human antibody DAW. For this reason, the FR of the human antibody DAW was used as a starting material for the production of the H chain V region of the reshaped human SK2 antibody.
[0047]
Two versions of the reshaped human SK2 antibody H chain V region were designed. In version “a”, the FR is the same as that of DAW, and in version “b” only the 30th position in human FR1 is different. That is, in version “a”, the amino acid at position 30 is serine, and the amino acid at position 30 in version “b” is asparagine.
Table 3 shows the amino acid sequences of versions “a” and “b” of mouse SK2 antibody H chain V region, DAW FR, and reconstituted SK2 antibody H chain V region.
[0048]
[Table 3]

[0049]
Production of reconstituted human SK2 antibody
The production of the reshaped human SK antibody V region is specifically described in Example 5.
The reconstituted human SK2 antibody of the present invention is
(A) (1) human L chain C region, and
(2) a light chain comprising a human light chain FR and a light chain V region comprising the light chain V region CDR of mouse monoclonal antibody SK2 against human IL-6; and
(B) (1) human H chain C region, and
(2) a heavy chain comprising a human heavy chain FR and a heavy chain V region comprising the heavy chain V region CDR of mouse monoclonal antibody SK2 against human IL-6;
Consists of
[0050]
In a preferred embodiment, the L chain V region has the amino acid sequence shown in SEQ ID NO: 52 or 53, and the H chain V region has the amino acid sequence shown in SEQ ID NO: 54 or 55. The human L chain C region can be any human L chain C region, eg, a human κC region; and the human H chain C region can be any human H chain C region, eg, human γ-1C region, human γ-4C region and the like.
[0051]
For the production of reconstituted antibodies, two types of expression vectors, ie expression comprising DNA encoding a reconstituted human light chain as defined above under the control of an expression control region such as an enhancer / promoter system. Another expression vector is made comprising a vector and DNA encoding a reshaped human heavy chain as defined above under an expression control region such as an enhancer / promoter system. These expression vectors are then used to cotransform host cells, such as mammalian cells, and the transformed cells are cultured in-vivo or in-vitro to produce reconstituted human antibodies (eg, , WO 91-16927).
[0052]
Alternatively, DNA encoding the reshaped light chain and DNA encoding the reshaped human heavy chain are introduced into a single expression vector and the vector is used to transform the host, which is then transformed into the transformed host. Cells are cultured in-vivo or in-vitro to produce the desired reconstituted human antibody.
The transformant transformed with the gene encoding the target chimeric antibody or humanized antibody is cultured as described above, and the produced chimeric antibody or humanized antibody is separated from the inside or outside of the cell and homogenized. Can be purified.
[0053]
In addition, separation and purification of the chimeric antibody or humanized antibody that is the target protein of the present invention can be performed using a protein A agarose column. In addition, any other separation / purification method used for ordinary proteins may be used, and the method is not limited at all. For example, the chimeric antibody or humanized antibody can be separated and purified by appropriately selecting and combining various types of chromatography, ultrafiltration, salt folding, dialysis and the like.
[0054]
Any expression system for the production of chimeric or reconstituted human antibodies of the present invention against human IL-6, such as eukaryotic cells, eg animal cells, eg established mammalian cell lines, filamentous fungal cells, and yeast cells , As well as prokaryotic cells such as bacterial cells such as E. coli cells. Preferably, the chimeric or reconstituted antibody of the invention is a mammalian cell, such asCOSExpressed in cells or CHO cells.
[0055]
In these cases, a conventional promoter useful for expression in mammalian cells can be used. For example, it is preferred to use the human cytomegalovirus immediate early (HCMV) promoter. Examples of expression vectors containing the HCMV promoter include HCMV-V_H-HCγ1, HCMV-V_L-HC_KAnd those derived from pSV2neo (see FIG. 2) (WO92-19759).
[0056]
In addition, examples of promoters for gene expression in mammalian cells that can be used for the present invention include viral promoters such as retrovirus, polyomavirus, adenovirus, and simian virus 40 (SV40), and human polypeptides. A mammalian cell-derived promoter such as chain elongation factor 1α (HEF-1α) may be used. For example, when the SV40 promoter is used, the method of Mulligan et al.277  108 (1979)), and when using the HEF-1α promoter, Mizushima, S .; (Nucleic Acids Research,18, 5322, 1990).
[0057]
As the origin of replication, those derived from SV40, polyoma virus, adenovirus, bovine papilloma virus (BPV), etc. can be used, and the expression vector is selected for amplification of the gene copy number in the host cell system. Markers may include phosphotransferase APH (3 ') II or I (neo) gene, thymidine kinase (TK) gene, E. coli xanthine-guanine phosphoribosyltransferase (Ecogpt) gene, dihydrofolate reductase (DHFR) gene, etc. it can.
[0058]
【Example】
Next, the present invention will be specifically described by the following examples, but the scope of the present invention is not limited thereby.
Example 1.Cloning of DNA encoding the V region of a mouse monoclonal antibody against human IL-6
DNA encoding the variable region of mouse monoclonal antibody SK2 against human IL-6 was cloned as follows.
[0059]
1.Preparation of total RNA
Total RNA from hybridoma SK2 was purchased from Chirgwin et al., Biochemistry,18, 5294 (1979). Briefly, hybridoma SK2 cells were completely homogenized in 20 ml of 4M guanidine thiocyanate (Fulka). The homogenate was overlaid on a 5.3 M cesium chloride solution layer in a centrifuge tube, and then this was centrifuged for 24 hours at 20 ° C. at 31,000 rpm in a Beckman SW40 rotor to precipitate the RNA.
[0060]
The RNA precipitate was washed with 80% ethanol and dissolved in 150 μl of 10 mM Tris-HCl (pH 7.5) containing 1 mM EDTA and 0.5% SDS, and Proteinase (Boehringer) was dissolved in 0.5 mg / ml. Then, the mixture was incubated at 37 ° C. for 20 minutes. The mixture was extracted with phenol and chloroform, and RNA was precipitated with ethanol. Next, the RNA precipitate was dissolved in 200 μl of 10 mM Tris-HCl (pH 7.5) containing 1 mM EDTA.
[0061]
2.Synthesis of single-stranded cDNA
J. et al. W. Larrick et al., Bio / Technology,7934 (1989), about 5 μg of total RNA prepared as described above was synthesized with 40 mM KCl, 6 mM MgCl.₂10 mM dithiothreitol, 0.5 mM dATP, 0.5 mM dGTP, 0.5 mM dCTP, 0.5 mM dTTP, 35 μM oligo dT primer (Amersham), 48 units of RAV-2 reverse transcriptase (RAV-2: Rous associated) virus2; Amersham) and 10 μl of 50 mM Tris-HCl (pH 8.3) buffer containing 25 units of human placental ribonuclease inhibitor (Amersham) and the reaction mixture is incubated at 37 ° C. for 60 minutes and then Used directly for the polymerase chain reaction (PCR) method.
[0062]
3.Amplification of gene encoding antibody variable region by PCR method
PCR was performed using Thermal Cycler Model PHC-2 (Techne).
(1)Amplification of gene encoding mouse L chain V region
Primers used in the PCR method are MKV (Mouse Kappa Variable) primers (ST Jones, et al., Bio / Technology, shown in SEQ ID NOs: 1 to 11 that hybridize with the mouse kappa type L chain leader sequence.9, 88, 1991), and the MKC (Mouse Kappa Constant) primer shown in SEQ ID NO: 12 (ST Jones, et al., Bio / Technology,9, 88, 1991).
[0063]
100 μl of PCR solution is 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 0.25 mM dNTPs (dATP, dGTP, dCTP, dTTP), 1.5 mM MgCl.₂  2.5 units of DNA polymerase AmpliTaq (Perkin Elmer Cetus), 0.25 μM of each MKV primer, 2.75 μM of MKC primer and 1 μl of reaction mixture for single-stranded cDNA synthesis. This was covered with 50 μl of mineral oil and then heated in this order for 1.5 minutes at an initial temperature of 94 ° C., then 94 ° C. for 1 minute, 50 ° C. for 1 minute and 72 ° C. for 1 minute. . After repeating this temperature cycle 25 times, the reaction mixture was further incubated at 72 ° C. for 10 minutes.
[0064]
(2)Amplification of cDNA encoding mouse H chain V region
As primers for PCR, MHV (Mouse Heavy Variable) primers 1-12 shown in SEQ ID NOs: 13-24, and MHC-G1 (Mouse Heavy Constant) primers (ST Jones Jones et al., Bio, shown in SEQ ID NO: 25). / Technology,9, 88, 1991).
[0065]
Amplification of cDNA was performed as described in 3 above, except that each MHV1-12 primer was amplified separately as a combination of 1 μM MHV primer and 1 μM MHC-G1 primer. Amplification was performed in the same manner as described for amplification of the L chain V region gene in (1).
[0066]
4).Purification and fragmentation of PCR products
The DNA fragment amplified by the PCR method as described above was purified with low melting point agarose (FMC Bio. Products, USA) and 10 mM MgCl 2₂  And 10 units of restriction enzyme SalI (manufactured by GIBCO BRL) in 100 mM Tris-HCl (pH 7.6) containing 150 mM NaCl for 3 hours at 37 ° C.
[0067]
The digestion mixture was extracted with phenol and chloroform and the DNA was recovered by ethanol precipitation. The DNA precipitate was then digested with 10 units of restriction enzyme XmaI (New England Biolabs) for 2 hours at 37 ° C., and the resulting DNA fragment was digested with 1.5% low melting point agarose (FMC Bio. Products, USA). ) Was used for agarose gel electrophoresis.
[0068]
A piece of agarose containing a DNA fragment about 450 bp long was cut and melted at 65 ° C. for 5 minutes, and an equal volume of 20 mM Tris-HCl (pH 7.5) containing 2 mM EDTA and 200 mM NaCl was added. The mixture was extracted with phenol and chloroform, and the DNA fragment was recovered by ethanol precipitation and dissolved in 10 mM Tris-HCl (pH 7.5) containing 1 mM EDTA.
[0069]
Thus, a DNA fragment containing a gene encoding the mouse kappa type L chain variable region and a DNA fragment containing a gene encoding the mouse H chain variable region were obtained. Each of the above DNA fragments has a SalI sticky end at its 5'-end and an XmaI sticky end at its 3'-end.
[0070]
5).Ligation and transformation
About 0.1 μg of pUC19 vector prepared by digesting about 0.3 μg of the SalI-XmaI DNA fragment comprising the gene encoding the mouse kappa type L chain V region prepared as described above with SalI and XmaI; , 50 mM Tris-HCl (pH 7.4), 10 mM MgCl₂  Ligation was performed in a reaction mixture containing 10 mM dithiothreitol, 1 mM spermidine, 1 mM ATP, 0.1 μg / ml bovine serum albumin and 2 units T4 DNA ligase (New England Biolabs) at 16 ° C. for 16 hours. .
[0071]
Next, 7 μl of the above ligation mixture was added to 200 μl of E. coli DH5α competent cells and the cells were left on ice for 30 minutes, 42 ° C. for 1 minute and again on ice for 1 minute. Subsequently, 800 μl of SOC medium (Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring Harbor Laboratory Press, 1989) was added, incubated at 37 ° C. for 1 hour, and then 2 × YT agar medium (Molecular Laboratory: Molecular Laboratory: Sambrook et al., Cold Spring Harbor Laboratory Press, 1989) was seeded with this E. coli and incubated overnight at 37 ° C. to obtain an E. coli transformant.
[0072]
The transformant was cultured overnight at 37 ° C. in 5 ml of 2 × YT medium containing 50 μg / ml ampicillin and from this culture, the alkaline method (Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring, Plasmid DNA was prepared according to Harbor Laboratory Press, 1989).
[0073]
The plasmid containing the gene encoding the mouse kappa type L chain V region derived from hybridoma SK2 thus obtained was named psk2-k2.
According to the same method described above, a plasmid containing a gene encoding the mouse H chain V region derived from hybridoma SK2 was constructed from the SalI-XmaI DNA fragment and pUC-SK2-V_HNamed.
[0074]
Example 2.Determination of DNA base sequence
The nucleotide sequence of the cDNA coding region in the plasmid is referred to as Sequenase.^TMThe determination was made using a Version 2.0 kit (US Biochemical, USA).
First, about 3 μg of the plasmid obtained as described above was denatured with 0.2N NaOH, annealed with a sequencing primer, and according to the instructions attached to the kit.³⁵Labeled by S-dATP.
[0075]
The labeled DNA is then electrophoresed on a 6% polyacrylamide gel containing 8M urea, after which the gel is fixed with 10% methanol and 10% acetic acid, dried and subjected to autoradiography. The base sequence was determined.
The base sequence of the gene encoding the L chain V region of the mouse SK2 antibody contained in the plasmid psk2-k2 is shown in SEQ ID NO: 26. In addition, plasmid pUC-SK2-V_HSEQ ID NO: 27 shows the base sequence of the gene encoding the H chain V region of the mouse SK2 antibody contained in.
[0076]
The plasmid pUC-SK2-V_HE. coli containing Escherichia coli DH5α (pUC-SK2V_H) As an FERM BP-4269 on April 21, 1993, based on the Budapest Treaty at the Institute of Biotechnology, Institute of Industrial Science (Tsukuba, Ibaraki 1-3).
[0077]
Example 3.Determination of CDR
The general structure of the L region and H chain V regions are similar to each other, with each of the four framework parts linked by three hypervariable regions, ie complementarity determining regions (CDRs). The amino acid sequence of the framework is relatively well conserved, while the amino acid sequence variability of the CDR region is extremely high (Kabat, EA, et al., “Sequences of Proteins of Immunological Interest” US Dept. Health. and Human Services, 1983).
[0078]
Based on these facts, the amino acid sequence of the variable region of the mouse monoclonal antibody against human IL-6 was applied to the antibody amino acid sequence database prepared by Kabat et al. Decided as shown.
[Table 4]

[0079]
Example 4.Confirmation of expression of cloned cDNA (production of chimeric SK2 antibody)
Production of expression vector
In order to produce a vector expressing a chimeric SK2 antibody, cDNA clones psk2-k2 and pUC-SK2-V encoding mouse SK2κ L chain and H chain V regions, respectively._HWas modified by the PCR method. And it introduce | transduced into the HEF expression vector (refer the said WO92-19759) (refer FIG. 1).
[0080]
The rear primer SK2K2B (SEQ ID NO: 28) for the L chain V region and the rear primer SK2H1S (SEQ ID NO: 30) for the H chain V region hybridize to DNA encoding the beginning of the leader sequence of each V region. Soybean and Kozak consensus sequences (Kazak, M. et al., J. Mol. Biol.196, 947-950, 1987) and a HindIII restriction site. The forward primer SK2K2A (SEQ ID NO: 29) for the L chain V region and the forward primer 64hch-A (SEQ ID NO: 31) for the H chain V region hybridize to the DNA sequence encoding the end of the J region. And designed to have a splice donor sequence and a BamHI restriction site.
[0081]
10 mM Tris-HCl (pH 8.3), 50 mM KCl, 250 μM dNTPs, 1.5 mM MgCl₂, 100 pmole of each primer, 100 ng of template DNA (psk2-k2 or pUC-SK2-V_H), And 100 μl PCR reaction mixture containing 5 u Ampli Taq enzyme with 50 μl mineral oil and after initial denaturation at 94 ° C., 94 ° C. for 1 min, 50 ° C. for 1 min, 72 ° C. The 1 minute cycle was repeated 30 times, and finally the incubation was performed at 72 ° C. for 10 minutes.
[0082]
The PCR product was purified using a 1.5% low melting point agarose gel, digested with HindIII and BamHI, and subcloned into the pUC19 vector (Yanishe-Perron et al., Gene (1985) 33: 103-109). After DNA sequencing, the plasmid containing the gene encoding the chain V region with the correct DNA sequence was transformed into pUC-SK2V_LNamed. Plasmid pUC-SK2V_LE. coli containing Escherichia coli DH5α (pUC-SK2V_L) As an FERM BP-4270 on April 21, 1993, based on the Budapest Treaty, at the National Institute of Biotechnology, Tsukuba City, Ibaraki Prefecture. A HindIII-BamHI fragment of H chain V region and L chain V region having the correct DNA sequence was excised, and the expression vector HEF-V_H-Gγ1 is introduced to obtain HEF-SK2h-gγ1, and for the L chain V region, HEF-V_L-Gk was introduced to obtain HEF-SK2k-gk.
[0083]
Furthermore, in the H chain V region, the serine at position 30 is conserved in the H chain of most antibodies, but since position 30 is asparagine in the mouse SK2 antibody H chain, the antigen of the chimeric SK2 antibody In order to examine the effect of this change on the binding activity, using HEF-SK2h-gγ1 as a template, the rear primer SK2H1S and the front primer SK2HM1B (SEQ ID NO: 32) and the front primer 64hch-A and the rear primer (SK2HM1) (SEQ ID NO: 33) ) Was used to change position 30 from asparagine to serine by PCR mutagenesis.
[0084]
The mutant H chain V region was subjected to PCR in the same manner as described above, using primers SK2H1S and SK2HM1B, or primers SK2HM1 and 64hch-A, and using plasmid HEF-SK2h-gγ1 as template DNA. After PCR amplification, the two PCR products were purified using a low melting point agarose gel and used for the second PCR.
[0085]
Incubate 98 μl of PCR mixture containing 1 μg each of the two first PCR products and 5 u Ampli Taq for 2 minutes at 94 ° C., 2 minutes at 50 ° C. and 5 minutes at 72 ° C., then 100 pmole each External primers (SK2H1S and 64hch-A) were added. The PCR mixture was covered with 50 μl mineral oil and a second PCR was performed under the same conditions as described above. The 412 bp DNA fragment was purified, digested with HindIII and BamHI, and the expression vector HEF-V_H-After insertion into gγ1 and sequencing, plasmid HEF-SK2h-NTS containing the correct sequence was obtained.
[0086]
Transfection into COS cells
In order to observe the transient expression of the chimeric SK2 antibody,COSTested in cells. HEF-SK2k-gk and either HEF-SK2h-gγ1 or HEF-SK2h-NTS are electroporated using a Gene Pulser device (manufactured by BioRad).COSCells were cotransformed. Each DNA (10 μg) was added 1 × 10 in PBS.⁷  In addition to 0.8 ml aliquots of cells / ml, pulses were given in a volume of 1,900 V, 25 μF.
[0087]
After a recovery period of 10 minutes at room temperature, the electroporated cells contain 10% γ-globulin free fetal calf serum with or without 2 μg / ml tunicamycin (Sigma) It added to the DMEM culture solution (made by GIBCO). After 72 hours of incubation, culture supernatants are collected, cell debris removed by centrifugation, and applied to a Protein A agarose column (Affi-Gel Protein A MAPSII kit, BioRad) equilibrated with 5 volumes of binding buffer did. The column was washed with 15 volumes of binding buffer and then eluted with 5 volumes of elution buffer. The eluate was concentrated and the buffer was changed to PBS using a microconcentrator (Centricon 100, Amicon).
[0088]
ELISA
An ELISA plate for measuring antigen binding was prepared as follows. A 96-well plate was obtained from a mouse monoclonal antibody MH166 (Matsuda, T. et al., Eur. J. Immunol.) That recognizes an epitope of human IL-6 that differs from the epitope recognized by SK2.18951-956, 1988), and after blocking, recombinant human IL-6 was added. Samples of the chimeric SK2 antibody were serially diluted and added to each well, followed by alkaline phosphatase-conjugated goat anti-human IgG antibody (Sigma). After incubation and washing, the substrate solution was added and then the absorbance at 405 nm was measured.
[0089]
For measurement of binding inhibition, an ELISA plate was prepared as follows. A 96-well plate was added to a mouse monoclonal antibody MT-18 (Hirata, Y. et al., J. Immunol., Specific for human IL-6 receptor (IL-6R).143, 2900-2907, 1989) and after blocking, soluble recombinant human IL-6R (SR344) (Yasukawa, K. et al., J. Biochem.108673-676, 1990). Samples were serially diluted and added to each well with biotinylated IL-6, then alkaline phosphatase-conjugated streptavidin was added, and the absorbance at 405 nm was measured as described above.
[0090]
The result of the antigen binding measurement is shown in FIG. 4, and the result of the binding inhibition measurement is shown in FIG. Asparagine is position 30 in the H chain V region. The chimeric antibody and the chimeric antibody (NTS) in which position 30 of the H chain V region was mutated to serine showed similar antigen binding and binding inhibitory activity without being affected by inhibition of glycosylation by tunicamycin. Therefore, it has been clarified that the mutation of the 30th amino acid in the H chain V region and the presence or absence of sugar chains have no effect on the antigen binding.
[0091]
Analysis of mouse SK2 antibody glycans using SDS / PAGE and Western blot
Fab fragments and Fc fragments were prepared from a mouse SK2 antibody using an Immuno Pure Fab Preparation kit (Pierce), and then a deglycosylated fragment was prepared using N-glycanase (Genzyme).
[0092]
These fragments were subjected to SDS-polyacrylamide gel electrophoresis and then stained with Coomassie Brilliant Blue (Coomassie Brilliant Blue, BioRad) or electrophoretically transferred to a nitrocellulose filter. In order to analyze the sugar added to the Fd fragment, the filter was applied to a panel of lectins (Lectin-Link, Genzyme).
[0093]
As shown in FIG. 3, the Fd fragment from the mouse SK2 antibody showed bands of about 34 kd and 33 kd in SDS / PAGE, and this ratio was about 1: 1. The Fd fragment deglycosylated with N-glycanase showed a molecular weight of approximately 29 kd, which was consistent with the value calculated from the amino acid composition of the Fd fragment. Also, the 34 kd Fd fragment and the 33 kd Fd fragment showed different reactivities to various lectins.
[0094]
That is, 34 kd Fd is Data Stramonium Agglutinin (DSA) +, Ricinus Communis Agglutinin (RCA) ++, Phaseolus Vulgaris Erythalinin (PHA-E) ++, Concanavalin The 33 kd Fd was DSA-, RCA +, PHA-E ++, ConA-, WCA-. These results revealed that the mouse SK2 antibody has at least two types of N-type sugar chains in the H chain V region.
[0095]
Example 5.Production of reconstituted human SK2 antibody
Production of reshaped human SK2 antibody L chain V region
Reconstituted human SK2 antibody L chain was prepared by CDR-grafting by PCR. This method is schematically shown in FIG. Eight PCR primers were used for the generation of reshaped human antibody SK2 (version “a”) with FRs derived from human antibody REI. External primers A (SEQ ID NO: 34) and H (SEQ ID NO: 35) were designed to hybridize with the DNA sequence of the pUC vector.
[0096]
CDR-grafting primer B (SEQ ID NO: 36), C (SEQ ID NO: 37) and D (SEQ ID NO: 38) have a sense DNA sequence, and CDR-grafting primer E (SEQ ID NO: 39), F (SEQ ID NO: 40) and G (SEQ ID NO: 41) have anti-sense DNA sequences and are complementary DNA sequences (15-35 bp) to the 5'-end DNA sequences of primers B, C and D, respectively. Have
[0097]
Four reactions AE, BF, CG, and DH were performed in the first PCR stage and each PCR product was purified. The four PCR products from the first PCR were assembled by their own complementarity (see WO92-19759). Next, external primers A and H were added to amplify the full length DNA encoding the reshaped human SK2 antibody L chain V region (second PCR). In the PCR, a plasmid pUC-RVL-PM1a-4 (WO92-19759) encoding a reshaped human PM-1 antibody L chain V region version “a” based on FR from human antibody REI was used as a template.
[0098]
In the first PCR step, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 250 μM dNTPs, 1.5 mM MgCl₂  100 μl of PCR mixture containing 100 ng template DNA, 100 pmole of each primer and 5 u of Ampli Taq was used. Each PCR tube was coated with 50 μl mineral oil. After first denaturing at 94 ° C., a reaction cycle of 94 ° C. for 1 minute, 50 ° C. or 55 ° C. for 1 minute and 72 ° C. for 1 minute was followed by incubation at 72 ° C. for 10 minutes.
[0099]
PCR products AE (187 bp), BF (87 bp), CG (152 bp) and DH (65 bp) were purified using a 1.5% low melting point agarose gel and assembled in a second PCR. did. In the second PCR, 1 μg of each first PCR product and 98 μl of a PCR mixture containing 5 u of Ampli Taq were added at 94 ° C. for 2 minutes, 50 ° C. for 2 minutes and 72 ° C. for 2 minutes. For 2 cycles and then 100 pmole of each external primer (A and H) was added. The PCR tube was covered with 50 μl of mineral oil and 30 cycles of PCR were performed under the same conditions as above.
[0100]
The 399 bp DNA fragment generated by the second PCR was purified on a 1.5% low melting point agarose gel, digested with BamHI and HindIII, and the resulting DNA fragment was HEF expression vector HEF-V._L-Cloned into gk. After DNA sequencing, the plasmid containing the DNA fragment encoding the correct amino acid sequence of version “a” of the reshaped human SK2 antibody L chain V region was designated HEF-RVL-SK2a. The amino acid sequence and base sequence of the L chain V region contained in this plasmid HEF-RVL-SK2a are shown in SEQ ID NO: 52.
[0101]
Version “b” of the reshaped human SK2 antibody L chain V region was generated by mutagenesis using PCR. Mutagen primers FTY-1 (SEQ ID NO: 42) and FTY-2 (SEQ ID NO: 43) were designed so that phenylalanine at position 71 was mutated to tyrosine. Plasmid HEF-RV1-SK2a was used as template DNA. The final PCR product was purified, digested with BamHI and HindIII, and the resulting DNA fragment was converted to HEF-expression vector HEF-V._LCloning into -gk gave plasmid HEF-RVL-SK2b. The amino acid sequence and base sequence of the L chain V region contained in this plasmid HEF-RVL-SK2b are shown in SEQ ID NO: 53.
[0102]
Production of reshaped human SK2 antibody H chain V region
A DNA encoding the reshaped human SK2 antibody H chain V region was designed as follows. The DNA sequence encoding the FR of human antibody DAW was designed based on the “codon usage” of the V region (Kabat, EA et al., US Dept Health and Human Services, US Government Printing Offices, 1991) and mouse SK2 A full-length DNA encoding version “a” of the reshaped human SK2 antibody H chain V region was designed by ligation with a DNA sequence encoding the CDR of the antibody H chain V region.
[0103]
Next, a HindIII recognition site / KOZAK consensus sequence and a BamHI recognition site / splice donor sequence were added to the 5′-side and 3′-side of the DNA sequence, respectively, so that they could be inserted into the HEF expression vector.
The DNA sequence thus designed was divided into 6 oligonucleotides and then computer analyzed for secondary structure in the oligonucleotides that could interfere with the assembly of these oligonucleotides.
[0104]
Six oligonucleotide sequences are shown in SEQ ID NOs: 44-49. These oligonucleotides are 93 to 96 bases long and have an overlap region of 20 to 24 bp. Three of the oligonucleotides, namely HP1 (SEQ ID NO: 44), HP3 (SEQ ID NO: 46) and HP5 (SEQ ID NO: 48) have a sense DNA sequence and the other three, namely HP2 (SEQ ID NO: 45), HP4 (SEQ ID NO: 47) and HP6 (SEQ ID NO: 49) have antisense DNA sequences. A method for assembling these six oligonucleotides by PCR is shown in FIG.
[0105]
98 μl of a PCR mixture containing 6 oligonucleotides in 2 pmoles each and 5 u Ampli Taq was first denatured at 94 ° C. for 2 minutes, followed by 94 ° C. for 1 minute, 50 ° C. for 1 minute and 72 Three cycles of incubation were performed consisting of 1 minute at 0 ° C followed by 10 minutes at 72 ° C. After adding 50 μmole each of HP1 and HP6 as external primers, the PCR tube was covered with 50 μl mineral oil and after the first denaturation at 94 ° C. for 1 min, 94 ° C. for 1 min, 50 ° C. for 1 min And 30 cycles of 1 minute at 72 ° C. and then incubated at 72 ° C. for 10 minutes.
[0106]
The 448 bp DNA fragment was purified using a 1.5% low melting point agarose gel, digested with HindIII and BamHI, and then HEF expression vector HEF-V_H-Cloned into gγ1. After DNA sequencing, the plasmid containing the DNA fragment encoding the correct amino acid sequence of the H chain V region was named HEF-RVH-SK2a. The amino acid sequence and base sequence of the H chain V region contained in this plasmid HEF-RVH-SK2a are shown in SEQ ID NO: 54.
[0107]
Version “b” was generated using mutagenesis by the PCR method. Mutagen primers RSK2-STNA (SEQ ID NO: 50) and RSK-STNB (SEQ ID NO: 51) were designed so that serine at position 30 was changed to asparagine. Plasmid RVH-SK2a was used as template DNA. The final PCR product is purified, digested with BamHI and HindIII, and then HEF expression vector HEF-V_H-Cloning into gγ1 yielded plasmid HEF-RVH-SK2b. The amino acid sequence and base sequence of the H chain V region contained in this plasmid HEF-RVH-SK2b are shown in SEQ ID NO: 55.
[0108]
In order to evaluate each chain of the reshaped human SK2 antibody, first, two expression vectors HEF-RVL-SK2a (version “a”) and HEF-RVL-SK2b (version “ b ") and the expression vector HEF-SK2h-gγ1 for the chimeric SK2 antibody H chain, and co-transfecting COS cells as described above and recovering the antibody product as described above, The antigen binding measurement was performed as described above.
[0109]
The result is shown in FIG. As shown in FIG. 8, a chimeric antibody (ChL / ChH) as a positive control, an antibody consisting of a reshaped L chain version “a” and a chimeric H chain (RVLa / ChH), and a reshaped L chain version “b” There is no difference in antigen binding between the antibody (RVLb / ChH) composed of a chimeric heavy chain and the change of the tyrosine residue to the phenylalanine residue at position 71 does not affect the antigen binding. It has been shown.
[0110]
Next, in order to evaluate the combination of two versions of the reshaped humanized SK2 antibody light chain and two versions of the reshaped humanized SK2 antibody heavy chain, HEF-RVL-SK2a and HEF-RVH-SK2a, HEF- Co-transfect COS cells with RVL-SK2a and HEF-RVH-SK2b, HEF-RVL-SK2b and HEF-RVH-SK2a, or HEF-RVL-SK2b and HEF-RVH-SK2b, and obtained as described above. Antigen binding measurements were performed on the antibodies obtained. The result is shown in FIG. As is clear from FIG. 9, all combinations of reshaped human H chain and reshaped human V chain showed good antigen binding comparable to the chimeric SK2 antibody.
[0111]
The plasmid HEF-RV_L-E. coli containing SK2b is Escherichia coli DH5α (HEF-RV_LSK2b), and plasmid HEF-RV_H-E. coli containing SK2a is Escherichia coli DH5α (HEF-RV_HSK2a) to the National Institute of Biotechnology, Tsukuba City, Ibaraki Pref. 1-3, Higashi 1-chome 1-3, Tsukuba City, Ibaraki Prefecture, on April 21, 1993 as FERM BP-4267 and FERM BP-4268, respectively. Based on the international deposit.
[0112]
In contrast, as shown in FIG. 10, in the measurement of binding inhibition (the ability of an antibody to inhibit binding between IL6 and IL-6R), the reshaped human SK2 antibody light chain version “b” (RvLb) and the reconstituted Only the combination with the human SK2 antibody heavy chain version “a” (RvHa) exhibits binding inhibitory activity comparable to the murine monoclonal antibody SK2 and the chimeric SK2 antibody, and this combination forms a functional antigen binding site in the human antibody. Was suggested.
[0113]
On the other hand, the combination of the reshaped human SK2 antibody light chain version “a” (RvLa) and the reshaped human SK2 antibody heavy chain version “a” (RvHa), and the combination of RvLa and RvHb exhibit low binding inhibitory activity, It was suggested that an antibody having phenylalanine at position 71 in the L chain shows good antigen-binding activity, but reforms a functional antigen-binding site.
Therefore, it can be said that tyrosine at position 71 is essential for reconstituting a functional antigen-binding site in the loop structure in the CDR1 region of the human antibody L chain.
[0114]
Reference Example 1  Production of hybridoma SK2
A hybridoma producing an anti-human IL-6 monoclonal antibody was prepared by fusing spleen cells of BALB / c mice immunized with human IL-6 and mouse myeloma cell line P3U1 by a conventional method using polyethylene glycol. Screening was performed using the activity of inhibiting the binding of IL-6 and IL-6R as an index, and 17 clones having binding inhibitory activity were obtained. These clones were transplanted into the abdominal cavity of a mouse, and the obtained ascites was applied to a protein A agarose column to obtain a purified mouse monoclonal antibody.
[0115]
Reference Example 2  Typing of mouse monoclonal antibody SK2
In order to examine the type of L chain and H chain of mouse monoclonal antibody SK2 produced from hybridoma SK2, typing was performed using a mouse monoclonal antibody isotyping kit (Amersham International plc). As a result, it was revealed that the SK2 antibody has a κ type light chain and a γ1 type heavy chain.
[0116]
[Sequence Listing]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]
SEQ ID NO: 26
Sequence length: 379
Sequence type: Nucleic acid
Number of strands: double strand
Topology: Linear
Sequence type: cDNA
origin
Organism name: Mouse
Direct origin
Clone: psk2-k2
Features: . 60 sig peptide
61. . 279 mat peptide
Array
[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162]

[0163]

[0164]

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0175]

[0176]

[0177]

[0179]

[0180]

[Brief description of the drawings]
FIG. 1 shows an expression plasmid HEF-V comprising the human EFI-α promoter / enhancer useful for the expression of light and heavy chains, respectively._L-Gk and HEF-V_H-Gγ1 is shown.
FIG. 2 shows an expression vector comprising the human cytomegalovirus (HCMV) promoter / enhancer system that is useful for expression of the antibody peptides of the present invention.
FIG. 3 is an electropherogram showing that the SK2 antibody is glycosylated.
FIG. 4 is a graph showing the results of an ELISA for confirmation of the ability of the chimeric antibody of the present invention to bind to human IL-6.
FIG. 5 is a graph showing the results of an ELISA for confirmation of the ability of the chimeric antibody of the present invention to inhibit binding of IL-6 to human IL-6.
FIG. 6 is a diagram of the creation of a first version (version “a”) of the reshaped human SK2 antibody L chain V region.
FIG. 7 is a diagram of the creation of the first version (version “a”) of the reshaped human SK2 antibody H chain V region.
FIG. 8 is a graph showing the ability of an antibody consisting of reshaped human L chain + chimeric H chain for binding to human IL-6.
FIG. 9 is a graph showing the ability of the four reshaped human SK2 antibodies of the present invention for binding to human IL-6 compared to that of a chimeric antibody.
FIG. 10 compares the ability of four reshaped humanized SK2 antibodies of the present invention to inhibit IL-6 binding to IL-6R compared to that of SK2 chimeric antibody and mouse monoclonal antibody SK2. It is a graph to show.

Claims (21)

(A) The following amino acid sequence (1):

Forming Ru light (L) chain variable from (V) region, and (b) a human L chain constant (C) region,
Forming Ru, reshaped human antibody L chain against human interleukin-6 from. (A) The following amino acid sequence (2):

Forming Ru heavy (H) chain V region from, and (b) a human H chain C region,
Reshaped human antibody H chain to human interleukin-6 Ru consists. (A) The following amino acid sequence (3):

Forming Ru H chain V region from, and (b) a human H chain C region,
Reshaped human antibody H chain to human interleukin-6 Ru consists. (1) (a) The following amino acid sequence (1):

Forming Ru light (L) chain variable from (V) region, and (b) a human L chain constant (C) region,
Ru consists L chain; and (2) (a) the following amino acid sequence (2):

Forming Ru heavy (H) chain V region from, and (b) a human H chain C region,
Ru consists of H chain;
Forming Ru, reshaped human antibody against human interleukin-6 from. (1) (a) The following amino acid sequence (1):

Forming Ru light (L) chain variable from (V) region, and (b) a human L chain constant (C) region,
Ru consists L chain; and (2) (a) the following amino acid sequence (3):

Forming Ru heavy (H) chain V region from, and (b) a human H chain C region,
Ru consists of H chain;
Forming Ru, reshaped human antibody against human interleukin-6 from. DNA encoding the reshaped human antibody L chain according to claim 1. DNA encoding the reshaped human antibody heavy chain according to claim 2. DNA encoding the reshaped human antibody heavy chain according to claim 3. A recombinant vector comprising DNA encoding the L chain according to claim 6. A recombinant vector comprising DNA encoding the H chain according to claim 7. A recombinant vector comprising DNA encoding the H chain according to claim 8. A recombinant vector comprising the DNA encoding the L chain according to claim 6 and the DNA encoding the H chain according to claim 7. A recombinant vector comprising the DNA encoding the L chain according to claim 6 and the DNA encoding the H chain according to claim 8. A transformant cotransformed with the recombinant vector according to claim 9 and the recombinant vector according to claim 10. A transformant cotransformed with the recombinant vector according to claim 9 and the recombinant vector according to claim 11. A transformant transformed with the recombinant vector according to claim 12. A transformant transformed with the recombinant vector according to claim 13. The method for producing a reshaped human antibody according to claim 4, comprising culturing the transformant according to claim 14 and collecting the reshaped human antibody from the culture. The method for producing a reshaped human antibody according to claim 5, comprising culturing the transformant according to claim 15 and collecting the reshaped human antibody from the culture. The method for producing a reshaped human antibody according to claim 4, comprising culturing the transformant according to claim 16 and collecting the reshaped human antibody from the culture. The method for producing a reshaped human antibody according to claim 5, comprising culturing the transformant according to claim 17 and collecting the reshaped human antibody from the culture.

Images